WO1999026644A1

WO1999026644A1 - Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation

Info

Publication number: WO1999026644A1
Application number: PCT/US1998/025390
Authority: WO
Inventors: Kathleen Rodgers; Gere Dizerega
Original assignee: University of Southern California USC
Current assignee: University of Southern California USC
Priority date: 1997-11-26
Filing date: 1998-11-24
Publication date: 1999-06-03
Anticipated expiration: 2000-05-26
Also published as: AU1706399A; EP1047441A1; JP2002507383A; CA2310852A1; WO1999026644A9

Abstract

The present invention fulfills a need in the art for methods that promote hematopoietic and mesenchymal stem and lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments and analogues thereof and AII AT2 type 2 receptor agonists.

Description

METHOD FOR PROMOTING HEMATOPOIETIC AND MESENCHYMAL CELL PROLIFERATION AND DIFFERENTIATION

Cross Reference

This application claims priority from U.S. Provisional Application No.

60/066,593 filed November 26, 1997.

Field of the Invention This present invention relates to methods for use in accelerating the proliferation and differentiation of hematopoietic and mesenchymal cells.

Background of the Invention

Bone marrow contains pluripotent stem cells that are capable of reconstituting either the hematopoietic system or a wide range of mesenchymal tissues. The mechanisms by which hematopoietic and mesenchymal stem cells produce a range of progenitor cell types are quite dissimilar.

The hematopoietic system is composed of a multitude of cell generations ranging from the terminally differentiated to very primitive hematopoietic progenitor cells (HPC). (Traycoff, et al., Experimental Hematology 24:299-306, 1996). HPC are pluripotent progenitor cells that possess the ability to terminally differentiate into hematopoietic lineage-specific progenitor cells (HLSPC). Hematopoiesis is an ongoing process, and therefore HPC must provide a continuous source of HLSPC, which in turn can differentiate into red cells, platelets, monocytes, granulocytes and lymphocytes. (Prockop, Science 276:71-74, 1997). HPC proliferate either by "self- renewal", to produce HPC-type progeny cells, or with accompanying differentiation, to produce HLSPC. (Traycoff, et al., supra).

HPC transplantation therapy has been successful for a variety of malignant and inherited diseases and also provides myelopoietic support for patients undergoing high-dose chemotherapy or radiotherapy. (Emerson, Blood 87:3082-3088, 1996). However, stem cell transplantation has been limited by several features. First, acquiring a sufficient quantity of stem cells to achieve benefit after transfusion requires either extensive, operative bone marrow harvests or extensive pheresis procedures. (Emerson, supra). Next, even under these circumstances, only a limited number of useful cells is obtained. Finally, mature blood cell regeneration after transfusion is slow, so that little direct therapeutic benefit is seen for periods of 1 to 3 weeks. (Emerson, supra).

The development of in vitro culture techniques for hematopoietic cells combined with technologies for isolating relatively pure populations of HPC and HLSPC has made possible their ex vivo expansion. (Alcom and Holyoake, Blood Reviews 10:167-176, 1996, which is incorporated by reference herein). Successful ex vivo expansion of HPC, both by self-renewal and proliferation with differentiation, promises many clinical benefits, such as reduction of the number and duration of leucapheresis procedures required for autologous transplantation, thus reducing the risk of disease contamination in the apheresis products. (Alcom and Holyoake, supra). Furthermore, ex vivo expansion may render inadequate HPC populations in peripheral blood and umbilical cord blood sufficient for autologous transplantation and adult allogeneic transplantation respectively. Finally, ex vivo expansion of HPC will greatly increase their utility as gene therapy vehicles. (Alcom and Holyoake, supra). Similarly, ex vivo expansion of HLSPC promises substantial clinical benefits, such as re-infusion of expanded populations of myeloid precursor cells to reduce the period of obligate neutropenia following autologous transplantation, the generation of natural killer cells for use in adoptive immunotherapy protocols, generation of megakaryocyte precursors to alleviate post-transplant-associated thrombocytopenia and more efficient generation of delivery systems for gene therapy. (Alcom and Holyoake, supra).

Human bone marrow, umbilical cord blood, and peripheral blood progenitor cells mobilized by chemotherapy and/or cytokine treatment have been shown to be effective sources of HPC for transplantation following the administration of high-dose therapy to treat malignancy. (Holyoake, et al., Blood 87:4589-4595, 1996). Whatever the source of hematopoietic cells, most studies have used cultured cell populations selected on the basis of HPC-specific surface antigens, such as CD34. These cells can be readily obtained by a number of techniques. (Alcom and Holyoake, supra). The results of several clinical trials using ex vivo expanded hematopoietic cells suggests that a fairly small number of HPC cultured ex vivo under appropriate conditions can initiate hematologic reconstitution. (Emerson, supra).

Survival and proliferation of HPC in ex vivo culture requires a combination of synergizing growth factors; the choice of cytokine/growth factor combination and culture system used will largely determine the fate of cells used to initiate the culture. (Alcorn and Holyoake, supra). In vivo, blood cell production is thought to be regulated locally by interactions of hematopoietic stem cells with a variety of cell- bound and secreted factors produced by adjacent bone marrow stromal cells. (Alcom and Holyoake, supra). The addition of growth factors and cytokines to the culture medium is intended to compensate for the absence of stroma-associated activities. Growth factors and cytokines that have been shown to increase production of HPC (in various combinations) include granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), and interleukins 1, 3, 6, and 11 (Reviewed in Takaku, J. Cancer Res. Clin. Oncol. 121:701-709, 1995; Holyoake, et

al., supra). Conversely, inclusion of macrophage inhibitory protein-lα (MlP-lα),

tumor necrosis factor α (TNF-α) or transforming growth factor β (TGFβ) in most

expansion cultures reported to date results in decreased HPC and HLSPC yields.

(Emerson, supra).

A great deal of effort has gone into defining the optimal conditions for ex vivo culture of hematopoietic cells. Methods that increase the ex vivo proliferation of HPC will greatly increase the clinical benefits of HPC transplantation. This is true both for increased "self-renewal", which will provide a larger supply of HPC capable of reconstituting the entire hematopoietic system, and for proliferation with differentiation, which will provide a larger supply of lineage-specific progenitor cells. Similarly, methods that increase in vivo proliferation of HPC will enhance the utility of HPC transplantation therapy by rapidly increasing local concentrations of HPC (and HLSPC) in the bone marrow. Furthermore, methods that result in the differentiation of HPC and HLSPC are useful in providing populations of specific cell types for use in cell therapy.

Transfection of mammalian HPC has been accomplished. (Larochelle, et al., Nature Medicine 2:1329-1337, 1996). Thus, methods that increase the proliferation of HPC and HLSPC are also useful in rapidly providing a large population of transfected cells for use in gene therapy.

Mesenchymal stem cells (MSC) are pluripotent progenitor cells that possess the ability to differentiate into a variety of mesenchymal tissue, including bone, cartilage, tendon, muscle, marrow stroma, fat and dermis as demonstrated in a number of organisms, including humans (Bruder, et al., J. Cellul. Biochem. 56:283-294 (1994). The formation of mesenchymal tissues is known as the mesengenic process, which continues throughout life, but proceeds much more slowly in the adult than in the embryo (Caplan, Clinics in Plastic Surgery 21 :429-435 (1994). The mesengenic process in the adult is a repair process but involves the same cellular events that occur during embryonic development (Reviewed in Caplan, 1994, supra). During repair processes, chemoattraction brings MSC to the site of repair where they proliferate into a mass of cells that spans the break. These cells then undergo commitment and enter into a specific lineage pathway (differentiation), where they remain capable of proliferating. Eventually, the cells in the different pathways terminally differentiate (and are no longer able to proliferate) and combine to form the appropriate skeletal tissue, in a process controlled by the local concentration of tissue-specific cytokines and growth factors (Caplan, 1994, supra).

Recently, it has been hypothesized that the limiting factor for MSC-based repair processes is the lack of adequate numbers of responsive MSC at the repair site (Caplan, 1994, supra). Thus, it has been suggested that by supplying a sufficient number of MSC to a specific tissue site the repair process can be controlled, since the repair site will supply the appropriate exposure to lineage-specific growth factors and differentiation molecules (Caplan, 1994, supra). Towards this end, several animal studies have demonstrated the feasibility of using autologous MSC for repair of various defects associated with mesenchymal tissue. (For review, see Caplan, et al., in The Anterior Cruciate Ligament: Current and Future Concepts, ed. D.W. Jackson, Raven Press, Ltd. NY pp.405-417 (1993). Recent work has demonstrated the feasibility of collection, ex vivo expansion in culture, and intravenous infusion of MSC in humans (Lazarus, et al., Bone Marrow Transplantation 16:557-564 (1995); Caplan and Haynesworth, U.S. Patent No. 5,486,359). Further, MSC of animal origin have been transfected with retroviruses and have achieved high level gene expression both in vitro and in vivo (Allay, et al., Blood 82:477A (1993). Thus, the manipulation of MSC via such techniques seems a promising tool for reconstructive therapies and may be useful for gene therapy.

MSC therapy can serve as a means to deliver high densities of repair- competent cells to a defect site when adequate numbers of MSC and MSC lineage- specific cells are not present in vivo, especially in older and/or diseased patients. In order to efficiently deliver high densities of MSC to a defect site, methods for rapidly producing large numbers of MSC are necessary. While MSC have been exposed to a number of growth factors in vitro, only platelet-derived growth factor (PDGF) showed mitotic activity (Caplan et al., 1994, supra), while none have been demonstrated to independently induce differentiation. Methods that increase the ex vivo proliferation and differentiation of MSC will greatly increase the utility of MSC therapy. Similarly, methods that increase in vivo proliferation and differentiation of MSC will enhance the utility of MSC therapy by rapidly increasing local concentrations of MSC at the repair site. Methods that allow for. ex vivo differentiation of MSC would also provide an important tool for cell therapy. MSCs from various species have been differentiated in vitro into colonies of osteoblasts, chondrocytes, and adipocytes in response to dexamethasone, 1,25 dihydroxyvitamin D₃, or BMP -2. (Prockop, Science 276:71-74, 1997) For example, ex vivo culturing of MSC to produce chondrocytes can be used to resurface joint cartilage in patients with degenerative arthritis or in reconstractive plastic surgery in patients with osteoarthritis. Similarly, treatment of MSC to differentiate into osteoclasts can be used for implantation into poorly healing bone.

Furthermore, methods that enhance the proliferation of lineage-specific descendants of MSC, including but not limited to bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage-specific cell types at appropriate repair sites.

Thus, there exists a need in the art for methods that increase the proliferation and differentiation of hematopoietic and mesenchymal pluripotent and lineage- specific cells that are useful in rapidly providing a large population of such cells for use in cell therapy and for making a large population of transfected cells for use in gene therapy.

Summary of the Invention

The present invention fulfills a need in the art for methods that promote hematopoietic and mesenchymal stem and lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All), All analogues, All fragments and analogues thereof and All AT₂ type 2 receptor agonists.

Brief Description of the Drawings Figure 1 is a graph showing the effect of All on the phagocytic capability of murine macrophages.

Figure 2 is a graph showing the effect of All on the phagocytic capability of rat macrophages.

Figure 3 is a graph showing the effect of All on respiratory burst function in rat peritoneal macrophages.

Figure 4 is a graph showing the effect of All on respiratory burst function in human

PBMC.

Figure 5 is a graph showing the effect of AII(l-7) (SEQ. ID. NO:4) on respiratory burst function in rat peritoneal macrophages. Figure 6 is a graph showing the effect of GSD 24B (SEQ ID NO:31) on respiratory burst function in rat peritoneal macrophages.

Figure 7 is a graph showing the effect of GSD 22 A (SEQ ID NO: 18) on respiratory burst function in rat peritoneal macrophages.

Figure 8 is a graph showing the effect of GSD 28 (SEQ ID NO:37) on respiratory burst function in rat peritoneal macrophages.

Figure 9 is a graph showing the effect of All on proliferation in response to pokeweed mitogen.

Figure 10 is a graph showing the effect of All on rat bone marrow cultures.

Figure 11 is a graph showing the effect of All on rat bone marrow cultures. Figure 12 is a graph showing the effect of All on murine HSC cultures.

Figure 13 is a graph showing the effect of All on murine HSC cultures.

Figure 14 is a graph showing the effect of All on murine HSC cultures.

Figure 15 is a graph showing the effect of All on murine HSC cultures. Figure 16 is a graph showing the effect of All on murine HSC cultures.

Figure 17 is a graph showing the effect of All on murine HSC cultures.

Figure 18 is a graph showing the effect of AII(l-7) (SEQ ID NO:4) on MSC proliferation.

Figure 19 is a graph showing the effect of GSD 22A (SEQ ID NO: 18) on MSC proliferation.

Figure 20 is a graph showing the effect of GSD 24B (SEQ ID NO:31) on MSC proliferation.

Figure 21 is a graph showing the effect of GSD 28 (SEQ ID NO:37) on MSC proliferation. Figure 22 is a graph showing the effect of All on alkaline phosphatase expression by

MSC.

Figure 23 is a graph showing the effect of All on GM-CSF secretion by mouse mesenchymal stem cells.

Detailed Description Of The Invention

Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A

Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press),

Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2" Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.).

As defined herein, the term "HPC" refers to any hematopoietic pluripotent progenitor cells capable of giving rise to a wide variety of differentiated hematopoietic cell types. Among cell types included within this definition are CD34⁺ bone marrow mononuclear cells (BMMC) (Berardi, et al, Blood 86:2123-2129, 1995), PBSC (Fritsch, et al, Bone Marrow Transplantation 17:169-178, 1996), cobblestone area forming cells (CAFC) (Lemieux, et al., Blood 86:1339-1347, 1995) and 5-FU BM cells (Alcom and Holyoake, Blood Reviews 10:167-176, 1996). As defined herein, the term "HLSPC" refers to hematopoietic lineage-specific progenitor cells, and includes the progeny of HPC that are committed to a cell-specific differentiation path. As defined herein, mesenchymal stem cells (MSC) are pluripotent progenitor cells that possess the ability to differentiate into a variety of mesenchymal tissue, including bone, cartilage, tendon, muscle, marrow stroma, fat and dermis. As defined herein, "proliferation" encompasses both cell self renewal and cellular proliferation with accompanying differentiation. As defined herein

"differentiation" refers to any cellular processes that distinguish a non-committed cell type from a more lineage committed cell type.

U.S. Patent No. 5,015,629 to DiZerega (the entire disclosure of which is hereby incoφorated by reference) describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of angiotensin II (All) in an amount which is sufficient for said increase. The application of All to wound tissue significantly increases the rate of wound healing, leading to a more rapid re-epithelialization and tissue repair. The term All refers to an octapeptide present in humans and other species having the sequence Asp-Arg-Nal-Tyr-Ile-His- Pro-Phe [SEQ ID ΝO:l]. The biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen. The substance so formed is a decapeptide called angiotensin I (AI) which is converted to All by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from AI. All is a known pressor agent and is commercially available.

Studies have shown that All increases mitogenesis and chemotaxis in cultured cells that are involved in wound repair, and also increases their release of growth factors and extracellular matrices (diZerega, U.S. Patent No. 5,015,629; Dzau et. al., J. Moi. Cell. Cardiol. 21:S7 (Supp III) 1989; Berk et. al., Hypertension 13:305-14 (1989); Kawahara, et al., BBRC 150:52-9 (1988); Naftilan, et al, J. Clin. Invest. 83:1419-23 (1989); Taubman et al., J. Biol. Chem 264:526-530 (1989); Nakahara, et al., BBRC 184:811-8 (1992); Stouffer and Owens, Circ. Res. 70:820 (1992); Wolf, et al., Am. J. Pathol. 140:95-107 (1992); Bell and Madri, Am. J. Pathol. 137:7-12 (1990). In addition, All was shown to be angiogenic in rabbit comeal eye and chick chorioallantoic membrane models (Fernandez, et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble, et al., Eur. J. Pharmacol. 195:305-6 (1991). Therefore, All may accelerate wound repair through increased neovascularization, growth factor release, reepithelialization and/or production of extracellular matrix. A peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) has been identified. This peptide is p- aminophenylalanine6-AII ["(p-NH₂-Phe)6- All)"] , Asp- Arg-Val-Tyr-Ile-Xaa-Pro-Phe

[SEQ ID NO.36] wherein Xaa is p-NH₂-Phe (Speth and Kim, BBRC 169:997-1006 (1990). This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al, Eur. J. Pharmacol. 256:93-97 (1994);

Bryson, et al., Eur. J. Pharmacol. 225:119-127 (1992).

The effects of All receptor and All receptor antagonists have been examined in two experimental models of vascular injury and repair which suggest that both All receptor subtypes (ATI and AT2) play a role in wound healing (Janiak et al.,

Hypertension 20:737-45 (1992); Prescott, et al., Am. J. Pathol. 139:1291-1296 (1991);

Kauffman, et al, Life Sci. 49:223-228 (1991); Viswanathan, et al, Peptides 13:783-

786 (1992); Kimura, et al., BBRC 187:1083-1090 (1992).

Many studies have focused upon AII(l-7) (All residues 1-7) or other fragments of All to evaluate their activity. AII(l-7) elicits some, but not the full range of effects elicited by AIL Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992);

Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J.

Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al, J. Pharmacol. Exper.

Ther. 265:664-673 (1991); Jaiswal, et al., Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111:652-654 (1994).

As hereinafter defined, a preferred class of AT2 agonists for use in accordance with the present invention comprises All, All analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AIL In addition to peptide agents, various nonpeptidic agents (e.g., peptidomimetics) having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.

The active All analogues, fragments of All and analogues thereof of particular interest in accordance with the present invention are characterized as comprising a

1 o sequence consisting of at least three contiguous amino acids of groups R -R in the sequence of general formula I

R^R^R^R^R^R^R^R⁸ in which R¹ and R² together form a group of formula X-R^A-R^B-, wherein X is H or a one to three peptide group and a peptide bond between R^A and R^B is labile to aminopeptidase A cleavage;

R is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr; R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homoSer and azaTyr;

R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Nal and Gly;

R⁶ is His, Arg or 6-ΝH₂-Phe;

R > 7 is Pro or Ala; and

R is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group. Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AH analogues set forth above subject to the restriction that R⁶ is p-NH₂-Phe.

In one class of preferred embodiments, R^A is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asp(NH ) and Sue. R^B is suitably selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys. Particularly preferred combinations for R^Λ and R^B are Asp- Arg, Asp-Lys, Glu- Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: AH, AIII or AH(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AH(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(l-7), Asp-Arg-Val-Tyr-Ile-His-Pro {SEQ ID NO:4]; AII(2-7). Arg-Val- Tyr-Ile-His-Pro [SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AH(5-8), Ile-His-Pro-Phe [SEQ ID NO:7]; AII(l-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8]; AH(l-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(l-4), Asp-Arg-Val- Tyr [SEQ ID NO:10]; and AH(l-3), Asp-Arg-Val [SEQ ID NO:l 1]. Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO: 12] and Arg- Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp- Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31]. AH(6-8), His-Pro-Phe [SEQ ID NO:14] and AH(4-8), Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.

Another class of compounds of particular interest in accordance with the present invention are those of the general formula II R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

in which R² is selected from the group consisting of H, Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃) , Thr, Ser, homo Ser and azaTyr;

R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val

and Gly; R⁶ is His, Arg or 6-NH₂-Phe;

R⁷ is Pro or Ala; and R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr.

A particularly preferred subclass of the compounds of general formula II has the formula

R²-R³-Tyr-R⁵-His-Pro-Phe [SEQ ID NO: 16]

wherein R , R and R are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His- Pro-Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO: 18]. The fragment AH(4-8) was ineffective in repeated tests; this is believed to be due to the exposed tyrosine on the N-terminus. In the above formulas, the standard three-letter abbreviations for amino acid residues are employed. In the absence of an indication to the contrary, the L-form of the amino acid is intended. Other residues are abbreviated as follows:

TABLE 1 Abbreviation for Amino Acids

It has been suggested that AH and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974). In general, it is believed that neutral side chains in position R³, R⁵ and R⁷ may be involved in maintaining the appropriate distance between active groups in positions R⁴, R⁶ and R⁸ primarily responsible for binding to receptors and/or intrinsic activity. Hydrophobic side chains in positions R³, R⁵ and R⁸ may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.

Appropriate side chains on the amino acid in position R² may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R².

For purposes of the present invention, it is believed that R³ may be involved in the formation of linear or nonlinear hydrogen bonds with R⁵ (in the gamma turn model) or R⁶ (in the beta turn model). R³ would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R may suitably be selected from Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr.

With respect to R⁴, conformational analyses have suggested that the side chain in this position (as well as in R and R ) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R⁴ is preferably selected from Tyr, Thr, Tyr (PO₃) , homoSer, Ser and azaTyr. In this position, Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).

In position R⁵, an amino acid with a β aliphatic or alicyclic chain is

particularly desirable. Therefore, while Gly is suitable in position R⁵, it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, Gly and Val.

In the AH analogues, fragments and analogues of fragments of particular interest in accordance with the present invention, R⁶ is His, Arg or 6-NH₂-Phe. The unique properties of the imidazole ring of histidine (e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R⁶. For example, conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel stmcture by influencing the orientation of R⁷. Similarly, it is presently considered that R⁷ should be Pro in order to provide the most desirable orientation of R⁸. In position R⁸, both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.

Analogues of particular interest include the following: TABLE 2

Angiotensin II Analogues

The polypeptides of the instant invention may be synthesized by any conventional method, including, but not limited to, those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis. The disclosures of the foregoing treatises are incoφorated by reference herein.

In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein incoφorated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.

Preferably, peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif), according to manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art. Alternatively, the peptides can be produced via recombinant DNA technologies. Techniques for recombinant production of the peptides of the invention are well known in the art and can be found in references such as Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA),

Although AH has been shown to increase the proliferation of a number of cell types in vitro, it does not necessarily increase the proliferation of all cell types. Studies have shown that AH accelerates cellular proliferation through the production

of transforming growth factor β (TGFβ) (Gibbons et al., J. Clin. Invest. 90:456-461

(1992). Thus, since only PDGF is known to be mitogenic for MSC, an ability of AH to effect MSC proliferation would be unexpected. Furthermore, as Emerson (supra)

has shown that inclusion of TGF-β in most expansion cultures resulted in a decreased

HPC and HLSPC yield, it is unexpected that AH, through the action of TGF-β, would

be of benefit in such situations. No studies have reported that AH has an effect on the differentiation of either HPC or MSC.

In one aspect of the present invention, a method of increasing ex vivo HPC and HLSPC proliferation by exposure to angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AH), AH analogues,

AH fragments and analogues thereof and AH AT₂ type 2 receptor agonists is disclosed. Experimental conditions for the isolation, purification, ex vivo growth and in vivo mobilization of HPC and HLSPC have been reported (Berardi, et al., Blood 86(6):2123-2129, 1995; Fritsch, et al., Bone Marrow Transplantation 17:169-178, 1996; LaRochelle, et al., Nature Medicine 12:1329-1337, 1996; Traycoff, et al., Experimental Hematology 24:299-306, 1996; Holyoake, et al., Blood 87:4589-4595, 1996; Lemieux, et al., Blood 86:1339-1347, 1995 ; Talmadge, et al., Bone Marrow transplantation 17:101-109, 1996; Bodine, et al., Blood 88:89-97, 1996; all references hereby incoφorated by reference herein.)

In one embodiment of the invention, HPC are isolated from bone marrow, peripheral blood or umbilical cord blood. HPC is then selected for in these samples. HPC-enriched samples are cultured under appropriate growth conditions, in the presence of angiotensin II (AH), AH analogues, AH fragments and analogues thereof and or AH AT₂ type 2 receptor agonists. HPC proliferation is assessed at various time points during culture.

In a preferred embodiment, HPC and HLSPC are isolated from bone marrow aspirates from the posterior iliac crest. CD34⁺ HPC are isolated from the aspirate by attaching a biotinylated monoclonal antibody specific for CD34 (available from Becton Dickinson, Sunnyvale, CA, USA) to a streptavidin affinity column (Ceprate SC; CellPro, Bothell, WA, USA) and passing the aspirate through the column, followed by appropriate column washing and stripping, according to standard techniques in the art. CD34⁺ HPC are suspended in culture medium and incubated in the presence of between 0.1 ng/ml and 1 mg/ml angiotensin II (AH), AH analogues,

AH fragments and analogues thereof and/or AH AT₂ type 2 receptor agonists. The cells are expanded for a period of between 8 and 21 days and cellular proliferation with accompanying differentiation is assessed via phase microscopy following standard methylcellulose colony formation assays (Berardi, et al., supra) at various points during this time period. Similarly, "self-renewal" of HPC is assessed periodically by reactivity to an antibody directed against CD34⁺.

In a further preferred embodiment, HPC that have been cultured in the presence of angiotensin II (AH), AH analogues, All fragments and analogues thereof and/or AH AT type 2 receptor agonists are used for autologous transplantation, to reconstitute a depleted hematopoietic system. Prior to transplantation, the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10⁶ and 50 x 10⁶ cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions. Following transplantation, subject peripheral blood samples are evaluated for increases in the number of HPC, HLSPC, and more mature blood cells at various time points by standard flow cytometry and cell sorting techniques. (Talmadge, et al., supra).

In another aspect of the present invention, a method of increasing ex vivo MSC and lineage-specific mesenchymal cell proliferation by exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists is disclosed. Experimental conditions for the isolation, purification and in vitro growth of lineage- specific mesenchymal cells, such as bone-marrow derived stromal cells, have been reported (Johnson and Dorshkind, Blood 68(6):1348-1354 (1986); hereby incoφorated by reference in its entirety). Other reports describe different conditions for culturing lineage-specific mesenchymal cells in vitro (Bab, et al., J. Cell Sci. 84:139-151 (1986); Benayahu, et al., J. Cellular Physiology 140:1-7 (1989); both references hereby incoφorated by reference in their entirety). In one embodiment of the present invention, MSC are isolated from bone marrow aspirates from the posterior iliac crest and/or femoral head cancellous bone, purified, resuspended in appropriate growth medium, counted and diluted to an appropriate concentration to seed in tissue culture plates. Purified MSC are cultured in an appropriate growth medium and growth conditions in a humidified atmosphere. The cells are allowed sufficient time to attach to the tissue culture dish, whereupon

non-attached cells are discarded. Adherent cells are placed in growth medium at 37°

C in a humidified atmosphere in the presence of between 0.1 ng/ml and 1 mg/ml angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and/or AH AT₂ type 2 receptor agonists. The cells are expanded for a period of between 2 and 21 days and cellular proliferation is assessed at various time points during this time period. Subsequent medium changes are performed as needed. When the primary cultures are nearly confluent, the cells are harvested for reinfusion into a subject. Cells are examined microscopically to verify the absence of contamination. The cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10⁶ and 50 x 10⁶ cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions. Subjects are evaluated for MSC proliferation in vivo by means of a repeat diagnostic bone marrow aspirate and biopsy to be compared with the original aspirate and biopsy. In a preferred embodiment, in vivo proliferation is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non- proliferating cells, such as proliferating cell nuclear antigen (PCNA, or cyclin). Such antibodies are commercially available from a number of sources, including Zymed Laboratories (South San Francisco, California).

In a preferred embodiment, isolated MSC are placed into Dulbecco's medium MEM (DMEM-LG) (Gibco, Grand Island, NY, USA). The cells are purified by a series of steps. Initially, the cells are pelleted and resuspended in complete medium. The cells are centrifuged through a 70% Percoll (Sigma, St. Louis, MO, USA) gradient at 460X g for 15 minutes, the top 25% of the gradients are transferred to a tube containing 30 ml of complete medium and centrifuged to pellet the cells, which will then be resuspended in complete medium, counted and diluted to seed in 100-mm plates at 50 x 10⁶ nucleated cells per plate.

In a further preferred embodiment, purified MSC are cultured in complete

medium at 37 °C in a humidified atmosphere containing 95% air and 5% CO₂ and the

cells are allowed to attach for 3 days, whereupon non-adherent cells are removed by changing the culture medium. Cellular proliferation of adherent cells and the presence of normal MSC moφhology are assessed by phase microscopy at various time points during the subsequent growth period. Subsequent medium changes are performed every four days. When the primary cultures are nearly confluent, the cells are detached with 0.25% trypsin containing 0.1 mM EDTA (Gibco) and either diluted and replated as second passage cells, or used for reinfusion into a subject. Preferably, cells are rinsed free of culture fluid using Tyrode's solution (Gibco). After the final

rinse, cells are placed in Tyrode's solution and placed in an incubator at 37 ° C for

one hour in order to shed serum proteins. The Tyrode's solution is removed and the cells are preferably placed into TCI 99 medium (Gibco) supplemented with 1% serum albumin. The cells are rinsed a number of times with this medium and after the final rinse MSC are resuspended in TCI 99 plus 1% serum albumin. Subsequently, MSC are injected slowly intravenously over 15 minutes. Evaluation of subsequent bone marrow aspirates are conducted up to 8 weeks after injection.

In a preferred embodiment, assessment of the in vivo proliferative effect of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists on MSC and mesenchymal lineage-specific cells is done by histochemical evaluations of various tissues. In a preferred embodiment, in vivo proliferation of MSC and mesenchymal lineage-specific cells is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA, or cyclin).

In a further aspect of the present invention, the effect of the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists on HPC, HLSPC, MSC and mesenchymal lineage-specific cell differentiation are assessed by examination of changes in gene expression, phenotype, moφhology, or any other method that distinguishes a cell undergoing differentiation from a progenitor cell. In one embodiment, MSC are incubated with angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT₂ type 2 receptor agonists as described above. MSC are then tested for the production of colony stimulating factors into the culture supernatant as a demonstration of MSC differentiation. In a preferred embodiment, the colony stimulating factor tested for is granulocyte-macrophage colony stimulating factor. In another preferred embodiment, macrophage differentiation to an elicited or activated state is assessed after exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT₂ type 2 receptor agonists, as described above. The macrophages are assessed for phagocytotic ability by any of the well known art methods, including but not limited to determination of the number of macrophages that have ingested opsonized yeast particles, and the number of yeast per macrophage ingested.

In another preferred embodiment, the respiratory burst activity of leukocytes is assessed after exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT₂ type 2 receptor agonists, as described above. The leukocytes are assessed for respiratory burst activity by any method known in the art, including but not limited to the ability to generate hydrogen peroxide via the respiratory burst system.

In another aspect of the present invention, angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT₂ type 2 receptor agonists are used to increase in vivo HPC, HLSPC, MSC and mesenchymal lineage-specific cell proliferation. For use in increasing proliferation of HPC, HLSCP, MSC and mesenchymal lineage-specific cells, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists may be administered orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrastemal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally. In a further embodiment of the present invention, a method of increasing in vivo HPC, HLSPC, MSC and lineage-specific mesenchymal cell proliferation by exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists is disclosed, either in the presence or absence of other growth factors and

cytokines.

The dosage regimen for increasing in vivo proliferation or differentiation of HPC, HLSCP, MSC and lineage-specific mesenchymal cell with angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. Dosage levels of the order of between 0.1 ng/ml and 100 mg/ml angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, angiotensin II (AH), AH analogues, AH fragments and analogues thereof and/or AH AT₂ type 2 receptor agonists are useful for all methods of use disclosed herein.

The angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT₂ type 2 receptor agonists may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.

While angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT₂ type 2 receptor agonists can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

For administration, the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT₂ type 2 receptor agonists are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, com oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

In a preferred embodiment of the present invention, the angiotensinogen, AI,

AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonist is administered topically.

A suitable topical dose of active ingredient of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, All analogues, AH fragments and analogues thereof and or AH AT type 2 receptor agonists is 1 mg/ml administered twice daily.

For topical administration, the active ingredient may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from

0.1% to 1%) of the formulation.

Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or

nose.

The present invention, by providing a method for enhanced proliferation of

HPC and HLSPC, will greatly increase the clinical benefits of HPC transplantation.

This is true both for increased "self-renewal", which will provide a larger supply of HPC capable of reconstituting the entire hematopoietic system, and for proliferation with differentiation, which will provide a larger supply of lineage-specific progenitor cells, for more rapid reconstitution of mature, functioning blood cells. Similarly, methods that increase in vivo proliferation of HPC will enhance the utility of HPC transplantation therapy by rapidly increasing local concentrations of HPC (and HLSPC) in the bone marrow, and thereby more rapidly producing functioning blood

cells.

Similarly, methods that increase the proliferation of MSC and mesenchymal lineage-specific cells, will greatly increase the utility of MSC therapy in the repair of skeletal tissues such as bone, cartilage, tendon and ligament. More rapid production of large numbers of MSC and mesenchymal lineage-specific cells will permit more efficient delivery of high densities of these cells to a defect site and more rapid in vivo amplification in the local concentration of stem and lineage-specific cells at an appropriate repair site. The method of the present invention also increases the potential utility of HPC and HLSPC as vehicles for gene therapy in hematopoietic disorders, as well as MSC and mesenchymal lineage-specific cells as vehicles for gene therapy in skeletal disorders by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected HPC, HLSPC, MSC and mesenchymal lineage-specific cells.

The present invention may be better understood with reference to the accompanying examples that are intended for puφoses of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended

hereto.

Example 1. Macrophage Differentiation: Phagocytosis

Resident peritoneal macrophages have very little phagocytic activity.

Exposure of macrophages to inflammatory or activating agents will increase this macrophage function. Resident peritoneal macrophages were harvested from C57B1/6 mice or Sprague Dawley rats and resuspended at a concentration of 1 x 10 cells/ml in phosphate buffered saline (PBS). Five separate 0.5 ml cell aliquots were placed on a glass coverslip in a 35 mm petri dish. Prior to incubation, either 0.5 ml of PBS, AH, or AH analogues or fragments at between 1-1000 ug/ml final concentration was added to the individual cover slips. The dishes containing the cover slips were then incubated at 37°C for 4 hours, after which the cover slips were washed 3 to 6 times with PBS. Opsonized yeast particles (Sigma Chemical Co.) (yeast opsonized with adult serum from the same species as that under study) were added to the cover slips and incubated for 2 hours, after which the cover slips were again washed with PBS and inverted onto a glass slide. The number of macrophages that ingested yeast and the number of yeast per macrophage ingested was then determined microscopically. At least 100 macrophages per coverslip were counted. The data from this study are summarized in Tables 3 and 4 and Figures 1 and 2. Table 5 describes the AH analogues and fragments used in these studies.

Table 3. Effect of All on the Phagocytic Capability of Murine Peritoneal Macrophages

Concentration of All (μg/ml)

0 1 10 100

1000

# Macrophages with Yeast

Yeast #

0 100 100 100 100 100

1-3 1 1 3 10 20

4-6 0 0 4 10 20

7-9 0 0 0 0 0

% Ingesting 1 1 6.5 16.7 25.6

# Yeast/MO 0.01 0.01 0.24 0.58 1.00 Table 4. Effect of AH on the Phagocytic Capability of Rat Peritoneal

Macrophages

Number of Macrophages with Yeast (Yeast Number) % w/Yeast #/Cell

1-3 4-6 >7

(Peptide) μg/ml

AII-0 0 0 2.2 0.04 100 90 30 20 0 35.7 1.14 1000 50 54 40 25 70.4 2.86

AII(l-7)-0 90 3 0 0 3.2 0.06 100 100 21 5 0 20.6 0.53 1000 90 30 10 0 30.8 0.85

GSD22A-0 90 1 0 0 1.1 0.02 100 100 26 5 0 23.7 0.59 1000 100 22 1 0 18.7 0.40

GSD 24-B-O 100 2 0 0 2.0 0.04 100 100 25 5 0 23.1 0.58 1000 100 10 0 0 9.1 0.18

GSD28-0 100 1 0 0 1.0 0.02 100 100 1 0 0 1.0 0.02 1000 100 1 0 0 1.0 0.02

Table 5. Designation for Analogues

Name Abbreviation Sequence SEQ ID NO:

GSD 28 Ile⁸-AII DRVYIHPI SEQ ID NO:37

GSD 24B Pro³-AII DRPYIHPF SEQ ID NO:31

GSD 22A Ala⁴-AIII RVYAHPF SEQ ID NO: 18

AII(l-7) DRVYIHP SEQ ID NO:4

Exposure to 10 μg/ml or greater AH tremendously increased the phagocytic

capability of peritoneal macrophages. Less than 1% of cells were phagocytic in the resident population (0.01 yeast per cell observed). After exposure to AH this increased to over 25% phagocytic at the highest concentration with on average 1 yeast observed per macrophage (25 fold increase in the number of macrophages able to phagocytose and a 100-fold increase in the number of particles phagocytized). As shown in Table 4, both concentrations of the peptides tested (with the exception of GSD 28) elevated that phagocytic capability of rat macrophages. However, none of the analogues resulted in the magnitude of an effect observed with AIL This suggests that AH and, to a lesser extent, AH analogues will stimulate macrophage differentiation to an elicited or activated state, which leads to the ingestion and clearance of bacteria and cellular debris.

Example 2 Leukocyte Differentiation: Respiratory Burst

The respiratory burst of leukocytes (macrophages and polymoφhonuclear neutrophils) is one component of the mediator system used to kill bacteria. As with phagocytosis, the level of this respiratory burst activity in resident macrophages is low. With differentiation, either to an elicited (inflammatory) or activated state, this functional activity is significantly elevated. Studies were conducted to assess the effect of in vitro exposure of murine or rat peritoneal macrophages and human peripheral blood mononuclear cells (PBMC) to various concentrations of AH on the

capacity to generate hydrogen peroxide via the respiratory burst system. For the human studies, five different donors were examined.

The murine or rat peritoneal cells were harvested by lavage with 5-15 ml of cold PBS with 0.5% bovine serum albumin. The human PBMC were harvested by venipuncture from normal human volunteers and isolated from peripheral blood by Ficoll Hypaque density centrifugation. After isolation, the cells were resuspended at

1 x 10⁶ cells/ml and placed at 100 μl per well into 96 well plates. The cells were

incubated with various concentrations of AH or All analogues and fragments for 4 hours at 37°C. The cells were then preloaded with a fluorescent probe for hydrogen peroxide, 2,7 dichlorofluorescein acetate, which is nonfluorescent in the absence of hydrogen peroxide. Fifteen minutes later, 10 ng/ml of phorbol myristate acetate (+PMA) or PBS (-PMA) was added to stimulate the production of hydrogen peroxide. One hour after stimulation the level of fluorescence produced was measured on a Cytofluor 2350 multiwell fluorometer. Representative results from this study are shown in Figures 3-8.

In the absence of PMA or peptide, no hydrogen peroxide production is observed. Some variability in the response to AH was seen (i.e. the concentration of AH necessary to increase the level of this function); however, in all studies AH increased the ability of leukocytes to generate hydrogen peroxide both alone and in response to stimulation with PMA. Further, the effect of pre-exposure to analogues and fragments of AH (GSD 22A, GSD 24B, GSD 28 and AH(l-7)) on the respiratory burst activity of PBMC was assessed (Figures 5-8). For all analogues and fragments, a much higher concentration of the peptide was needed to increase the respiratory burst activity. Up to 100 times more of these analogues were necessary; however, an increase in the respiratory burst capacity was observed for all analogues tested. The analogues were able to stimulate that function both in the presence and absence of PMA. These data indicate that AH was able to stimulate the differentiation of monocytes/macrophages from three species.

Example 3. Proliferative response of human lymphocytes

Upon stimulation of lymphocytes with mitogen or antigen, these cells undergo blastogenesis and proliferation. In the absence of such stimuli, proliferation is seldom observed. One method to measure cellular proliferation in a short term assay is via measurement of the amount of the nucleotide thymidine that is incoφorated into newly synthesized DNA. The effect of AH on the proliferation of human PBMC in the presence and absence of pokeweed mitogen ("PWM") was assessed.

Human PBMC were collected from normal volunteers and isolated via Ficoll Hypaque (Sigma Chemical, St. Louis) density centrifugation. After isolation of the buffy coat, the cells were washed 3X to remove the Ficoll Hypaque, counted in trypan blue (0.01%) and resuspended at a concentration of 1 x 10⁶ cells/ml in RPMI 1640

containing 10% fetal calf serum and antibiotics. A 100 μl aliquot of cells was added

to each well. Thereafter, various concentrations (0.1 to 1000 μg/ml final

concentration) of AH in RPMI 1640 containing 10% FCS and antibiotics were added

to various wells in triplicate. To the appropriate wells, PWM (20 μg/ml final

concentration) was added. These plates were incubated at 37°C in 5% CO for 48

hours. Subsequently, 0.5 μCi of H-thymidine was added to each well, which were

incubated at 37°C for an additional 24 hours prior to harvest by a multiwell automated sample harvester (Skatron) onto glass fiber filters. These filters were dried, placed in scintillation fluid and the amount of thymidine incoφorated was determined by beta counting. The results are shown in Figure 9.

In the absence of mitogen, no increase in thymidine incoφoration was observed after exposure to AIL However, in two separate experiments (cells from 2 different donors) AH was shown to increase the amount of thymidine incoφorated 50% to 100% in response to PWM. These data show that AH is able to increase the proliferation of cells from the hematopoietic lineage (e.g. lymphocytes). Example 4 Rat Mesenchymal Stem Cell Proliferation

These studies were conducted to determine the effect that AH would have on the proliferation of MSC. Bone marrow cells were harvested from the femur and tibia of female Sprague Dawley rats by flushing the bones with Dulbecco's Minimal Essential Medium-High Glucose ("DMEM-HG") with a syringe and an 18 gauge needle. These cells were cultured in 24 well plates at 5 x 10³ cells/mm² in DMEM- HG containing selected lots of fetal calf serum (FCS) and antibiotics (complete medium) at 37°C incubator containing 5% CO in air. Twenty-four hours after the initiation of the cultures the medium and nonadherent cells were aspirated and fresh medium was added. To each of these several wells, complete medium with (1 to 100

μg/ml) or without AH was added. The migration of cells from the clones and their

proliferation was followed by microscopic examination. Every 4 days the old medium was removed and fresh medium was added to the cultures.

The data from these experiments are shown in Figures 10 and 11. Addition of AH to the cultures significantly increased the number of sites from which the MSC were migrating (CFU) and the size (number of cells) of the colonies formed. This occurred only in the presence of serum that in itself would support MSC growth, albeit to a lesser extent. As can be seen, AH caused an increase in the number of MSC in a concentration dependent manner at all time points examined in the presence of two different serum lots. These data support the hypothesis that AH can increase the proliferation of rat MSC. Example 5 Effect of Angiotensin II on Murine Progenitor Cells

HPC were harvested from C57B1/6 mice by immunomagnetic affinity chromatography and placed in semi-solid cultures with optimal growth medium. At various times after initiation of culture, the formation of colonies and the size of the colonies (number of cells/colony) were assessed microscopically.

Female C57B1/6 mice were purchased from Simonson and used as a source of bone marrow cells in this study. The bone marrow was harvested, either from untreated mice or from mice injected with 5-fluorouracil (5-FU) (3 mg/mouse; approximately 150 mg/kg) in the tail vein 48 hours before harvest, from the femur and tibia of mice by flushing with phosphate buffered saline (PBS), pH 7.4, containing 2% fetal bovine serum (FBS) with a 21 -gauge needle. The eluant from the flushing was centrifuged and the pellet was resuspended at 4 x 10⁷ nucleated cells/ml in PBS containing PBS containing 2% FBS and 5% normal rat serum.

The reagents for immunomagnetic labeling were purchased from Stem Cell Technologies, Inc. (Vancouver, BC). Biotin-labeled monoclonal antibodies to the following murine lineage-specific cell surface antigens were included in a cocktail for HPC enrichment and used according to the manufacturer's instructions: CD5 (Ly-1), CD45-R (B220), CDl lb (Mac-1), Myeloid Differentiation Antigen (Gr-1) and

Erythroid Cells (TER 119) Ten μl of antibody cocktail was added for each of the 2

sets of bone marrow (normal and 5-FU-treated), mixed and allowed to incubate at 4°C

for 15 minutes. The cells were then resuspended at 4 x 10⁷ cells/ml in PBS containing

2%> FBS. The antibody cocktail was then washed out and 100 μl anti-biotin tetramer

was added for each ml of cells. The suspension was mixed and incubated at 4°C for 15 minutes. Sixty μl of magnetic colloid was then added for each ml of cells, the

combination was mixed and incubated at 4°C for 15 minutes to yield the

immunomagnetically-labeled bone marrow cells.

A column containing a stainless steel matrix was prepared by washing the matrix with PBS followed by washing with PBS containing 2% protein. The immunomagnetically-labeled bone marrow cells were loaded onto the column and unlabeled cell-containing medium (enriched HPC) was collected in the flow through

fraction at a flow rate of 0.2 ml/minute. Medium was added to the top of the column so that it did not run dry until 8 to 10 ml of enriched HPC were harvested. Approximately 2% of the cells loaded onto the column were isolated in the enriched HPC fractions.

The enriched HPC cell fractions were diluted into a semi-solid medium containing 0.9% methylcellulose in alpha minimal essential medium (alpha MEM), 30%) fetal calf serum, 1% bovine serum albumin, 10^"4 M 2-mercaptoethanol, 2 mM L- glutamine, and 2% conditioned medium containing colony stimulating factors. The conditioned medium was supernatant from splenocyte cultures (1 x 10⁶ cells/ml)

incubated for 48 hours in RPMI 1640 containing 10 μg/ml pokeweed mitogen

(PWM), 10% FCS, and antibiotics. Various concentrations of AH, between 0 and 100

μg/ml were added in a small volume to the wells of microtiter dishes, to which

between 2 x 10⁴ cells/ml for the normal and 2.5 x 10⁴ cells/ml for the 5-FU treated

cells. The cells were incubated at 37°C and 5% CO₂ for 14 days. At day 14 only,

macroscopic cell colonies were observed in the wells containing enriched HPC from

untreated (normal) mice treated with 10 μg/ml (18 macroscopic colonies) and 100 μg/ml AH (100 macroscopic colonies). Microscopic evaluation of the cells was

performed at various days after initiation of incubation, and the results are summarized in Figures 12-17.

Figures 12-14 and 16 represent the number of colonies containing more than a certain number of cells/colony as a function of the duration and concentration of AH exposure (Figures 12-14 for normal cells; Figure 16 for 5-FU treated cells.) Figures 15 and 17 represent the number of cells per colony seen after incubation of from normal (Figure 15) or 5-FU treated (Figure 17) mice with various concentrations of AH as a function of time. The results clearly demonstrate that HPC colony size increases proportionately with exposure to increased concentrations of AH, and thus that AH increases HPC proliferation.

Example 6. Effect of All Analogues and Fragments on Rat Mesenchymal Stem Cell Proliferation These studies were conducted to determine the effect that inclusion of AH analogues and fragments in the cell culture of MSC would have on the proliferation of these cells. Bone marrow cells were harvested from the femur and tibia of female Sprague Dawley rats by flushing the bones with Dulbecco's Minimal Essential Medium-High Glucose ("DMEM-HG") with a syringe and an 18 gauge needle. These cells were cultured in 24 well plates at 5 x 10³ cells/mm² in DMEM-HG containing selected lots of fetal calf serum (FCS) and antibiotics (complete medium) at 37°C incubator containing 5% CO in air. Twenty-four hours after the initiation of the cultures the medium and nonadherent cells were aspirated and fresh medium was

added. To each of these several wells, complete medium with (1 to 100 μg/ml) or without AH analogues and fragments (see Table 5) was added. The migration of cells from the clones and their proliferation was followed by microscopic examination. Every 4 days the old medium was removed and fresh medium was added to the cultures. Addition of AH analogues or AH fragments to these cultures had a profound effect on the number of sites from which MSC were migrating (CFU) and the size (number of cells) of the colonies formed. The results from these studies can be seen in Figures 18-21. As can be seen, AH analogues and fragments caused an increase in the number of MSC at all time points examined. These data indicate that AH analogues and fragments can increase the proliferation of rat MSC.

Example 7. Differentiation of MSC that have undergone proliferation in the presence ofAII. Mesenchymal stem cells isolated from bone marrow and grown under appropriate conditions can express characteristics of multiple cell types, including cells involved in the generation of bone, cartilage, muscle and tendons. Osteogenic cells (cells that can form bone tissue) express the enzyme alkaline phosphatase when cultured in medium that drives them toward their osteogenic differentiation. Bone marrow from female Sprague Dawley rats were harvested by flushing the femur with medium. The cells were placed in culture dishes 9 cm² in diameter, allowed to adhere overnight, and then placed in DMEM-LG medium containing antibiotics and 10% fetal calf serum together with varying concentrations of AIL At various times after culture initiation, the cells were washed with Tyrode's buffer and placed in osteogenic medium (DMEM-LG containing 10% fetal calf serum, 100 nM dexamethasone and 0.05 mM ascorbic acid) for 4 days prior to assessment of the level of alkaline phosphatase activity per well. Briefly, the wells were washed with Tyrode's buffer and 1 ml alkaline phosphatase substrate solution (50 mM glycine, pH 10.5, containing 1 mM magnesium chloride and 2.5 mM p-nitrophenyl phosphate) to each well. Fifteen minutes after addition of this aqueous substrate, the buffer was removed from the culture and mixed with 1 ml of IN sodium hydroxide to stop the reaction. The absorbance of the resultant mixture at 405 nm was then determined via spectroscopy. The level of alkaline phosphatase activity is expressed as the level of absorbance per culture dish. These data are shown in Figure 22 and demonstrate that AH can accelerate the proliferation of cells that express alkaline phosphatase when placed in medium appropriate to induce osteogenic differentiation.

Example 8. Induction of mesenchymal stem cell differentiation by All

Studies have also shown that exposure of murine bone marrow stem cells to AH increases the formation of colonies in the absence of exogenous colony stimulating factors. As these factors are necessary for the formation of colonies, these data suggested that AH may have stimulated non-hematopoietic cells (i.e.: mesenchymal cells) to release the necessary colony stimulating factors. This was further confirmed by data from cultures of further purified hematopoietic progenitor cells. After further purification of lineage-negative bone marrow cells to those that express the protein Seal, which eliminates mesenchymal cells from the culture, AH increased the number and size of colonies formed only in the presence of exogenous colony stimulating factors (U.S. Application Serial No. 09/012,400, hereby incoφorated by reference in its entirety). One factor whose production is induced during differentiation of mesenchymal stromal cells is granulocyte-macrophage-colony stimulating factor (GM-CSF). Therefore, the effect of AH on the differentiation of mesenchymal cells was assessed by the production of colony stimulating factors, specifically GM-CSF, into the culture supernatant.

Bone marrow cells were harvested from the femur and tibia of C57B1/6 mice (Battin and Kingdom, Gilroy, CA) with saline containing 2% fetal calf serum. Thereafter, the red blood cells were lysed by brief exposure to a hypotonic ammonium chloride solution. The nucleated cells were then resuspended in 3 ml of 5X phosphate buffered saline (PBS; pH 7.2) and rapidly layered over 7 ml of PercoU (70% PercoU gradient separation). This combination of nucleated bone marrow cells and PercoU were centrifuged at 1800 φm for 20 minutes. After centrifugation, the cells in the upper 25% of the PercoU gradient were harvested and washed 3 times with PBS to remove excess PercoU. The cells were then resuspended at 4 x 10⁵ cells/ml in alpha minimal essential medium containing 10% horse serum, 10% fetal calf serum and 5 x 10^"5 M 2-mercaptoethanol. These cells were cultured overnight in 24-well plates to allow adherence to the tissue culture plastic. Thereafter, the non-adherent cells were removed by washing with PBS and the medium was replaced with fresh medium containing various concentrations of angiotensin II (AH) (0.01- 100 μg/ml). Twenty four, 48, or 72 hours after addition of AH, supernatants were harvested and frozen until the time of assay.

After all samples were collected, the level of GM-CSF in the supernatant was measured by Quantikine ELISA (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. As shown by the data shown in Figure 23, exposure to

Claims

AH at concentrations ranging from 0.01 to 100 μg/ml induced an increase in the production of GM-CSF by murine mesenchymal stem cells. These data demonstrate that AH can induce mesenchymal stem cell differentiation.We claim:

1. A method of accelerating the differentiation of mesenchymal stem and lineage-specific cells comprising contacting the mesenchymal stem and lineage- specific cells with an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence

1 8 consisting of at least three contiguous amino acids of groups R -R in the sequence of general formula I

R¹ -R²-R³-R⁴-R⁵-R⁶-R^7"R⁸ in which R and R together form a group of formula X-R^A-R^B-, wherein X is H or a one to three peptide group and a peptide bond between R^A and R is labile to aminopeptidase A cleavage;

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He. Gly, Pro, Aib, Acpc and Tyr;

R⁵ is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;

R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group.

2. The method of claim 1 wherein the active agent is selected from the group consisting of AH, AIII, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ro NO:6, SEQ ro NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:31, and SEQ ID NO: 37.

3. The method of claim 1 wherein the concentration of active agent is between about 0.01 ng/kg and about 10.0 mg/kg.

4. An improved cell culture medium for growth of mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R*-R⁸ in the sequence of general formula I R'-R^-R^R-'-R^-R⁸

1 in which R and R together form a group of formula

X-R^A-R^B-, wherein X is H or a one to three peptide group and a peptide bond between R^A and R^B is labile to aminopeptidase A cleavage; R is selected from the group consisting of Val, Ala, Leu, norLeu, He,

Gly, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homoSer and azaTyr; R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val

and Gly;

5. The improved cell culture medium of claim 5 wherein the active agent is selected from the group consisting of AH, AIH, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:31, and SEQ ID NO: 37.

6. The improved cell culture medium of claim 4 wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.

7. A kit for the differentiation of mesenchymal stem and lineage-specific cells,

comprising:

(a) an amount effective to accelerate differentiation of mesenchymal stem and lineage- specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R'-R⁸ in the sequence of general formula I: R'-R²-R³-R⁴-R⁵-R⁶-R^7"R⁸ in which R and R together form a group of formula

X-R^A-R^B-, wherein X is H or a one to three peptide group and a peptide bond between R^A and R^B is labile to aminopeptidase A cleavage; R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homo Ser and azaTyr; R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val

and Gly;

R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group; and

(b) instructions for using the amount effective of active agent as a cell culture medium supplement.

8. The kit of claim 7 further comprising cell growth medium.

9. The kit of claim 7 further comprising a sterile container.

10. The kit of claim 7 wherein the active agent is selected from the group consisting of AH, AIH, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, and SEQ ID NO: 37.

11. The kit of claim 7 wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.

12. A method of accelerating the differentiation of mesenchymal stem and lineage-specific cells comprising contacting the mesenchymal stem and lineage- specific cells with an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R²-R⁸ in the sequence of general formula II

R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He,

Gly, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(PO )₂, Thr, Ser, homoSer and azaTyr; R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val

and Gly;

R° is His, Arg or 6-NH₂-Phe;

R is Pro or Ala; and

R is selected from the group consisting of Phe, Phe(Br), He and Tyr.

13. The method of claim 12 wherein the active agent is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:17, and SEQ ID NO:18.

14. The method of claim 12 wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.

15. An improved cell culture medium for growth of mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R >2 -R in the sequence of general formula II

R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

in which R is selected from the group consisting of H, Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;

and Gly;

R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and

R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr.

16. The improved cell culture medium of claim 15 wherein the active agent is selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 17, and SEQ ID

NO:18.

17. The improved cell culture medium of claim 15 wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.

18. A kit for the differentiation of mesenchymal stem and lineage-specific cells, comprising:

(a) an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R²-R⁸ in the sequence of general formula II

R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

and Gly;

R° is His, Arg or 6-NH₂-Phe;

R⁷ is Pro or Ala; and

R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr; and

19. The kit of claim 18 further comprising cell growth medium.

20. The kit of claim 18 further comprising a sterile container.

21. The kit of claim 18 wherein the active agent is selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 17, and SEQ ID NO: 18.

22. The kit of claim 18 wherein the concentration of active agent is between about

0.01 ng/ml and about 10.0 mg/ml.