CN102755638A - Application of AGGF1 in preparing medicines for promoting angiogenesis - Google Patents

Abstract

The invention relates to application of a growth factor AGGF1 in preparing medicines for promoting angiogenesis. The AGGF1 can be AGGF1 protein, and nucleotide sequence for coding AGGF1 or active segment thereof. The invention relates to a non-therapeutic method for promoting angiogenesis, which is implemented by giving effective vessel stimulation dose of AGGF1 protein or active segment thereof, and/or nucleotide segment for coding the AGGF1 protein or active segment thereof.

Description

Use of AGGF1 in the preparation of drugs for promoting angiogenesis

field of invention

The present invention relates to a method for treating cardiovascular system and its diseases by acting on skeletal muscle and cardiac muscle, and more specifically relates to a method of using growth factors to stimulate the proliferation and growth of endothelial cells, thereby stimulating angiogenesis to treat cardiovascular diseases Methods. The present invention also relates to the use of growth factor AGGF1 in the preparation of drugs for promoting angiogenesis, wherein the AGGF1 can be AGGF1 protein, nucleotide sequence encoding AGGF1 or active fragments thereof.

Background technique

Peripheral vascular disease, especially vascular disease of the lower extremities, is usually caused by narrowing or occlusion of blood vessels caused by atherosclerosis, thereby preventing blood flow to the lower extremities (J Am Coll Cardiol47: 1239-1312 (2006); Circulation 94: 3026- 3049 (1994)). In today's society, especially in the era of increasing aging, the incidence of lower extremity vascular diseases is also increasing, and the incidence rate of adults has reached 12% (J Am Coll Cardiol58: 2020-2045 (2011); New N Engl J Med 344:1608-1621 (2001)). There are approximately 8 to 12 million patients with lower extremity vascular disease in the United States (Circulation 110:738-743 (2004)). In Germany, the prevalence rate of women over 65 years old even reached 17%, and the prevalence rate of men was even higher, reaching 20% (Diabetes Care (Diabetes Care) 26: 3357-3358 (2003)).

There are also many clinical symptoms of lower limb ischemic disease, and some present typical symptoms, such as indirect claudication, but in many cases, patients with lower limb ischemic disease show other symptoms (J AmColl Cardiol 47:1239-1312( 2006); International Journal of Epidemiol (IntJ Epidemiol) 25: 1172-1181 (1996)). In some severe patients, lower extremity ischemia seriously affects quality of life and leads to high morbidity and mortality (New England Journal of Medicine (N Engl J Med) 326: 381-386 (1992)). The one-year survival rate of patients with severe lower extremity ischemia is close to 25%. More importantly, 46% of patients with lower extremity ischemia also suffer from coronary artery disease, and more than 10% of patients also have cerebrovascular disease (J Clin Epidemiol 45: 529-542 (1992); Circulation (Circulation) 122:1862- 1875 (2010)).

The current treatment of lower extremity vascular diseases mainly focuses on reducing the morbidity and mortality of cardiovascular and cerebrovascular diseases, while alleviating the symptoms of lower extremity vascular diseases Diabetes treatment (Mayo Clin Proc) 85(7): 678-692 (2010)). Lipid-lowering drugs. Drugs such as blood pressure-lowering drugs and antithrombotic drugs are used to prevent diseases such as myocardial infarction and cerebral infarction (Mayo Clin Proc 85(7): 678-692 (2010)). However, some patients with lower extremity vascular disease may suffer from great pain and have to choose amputation. Surgical procedures such as angioplasty, stenting, and peripheral vascular bypass are often used to reperfuse blood flow in the legs to avoid amputation and other serious conditions (Thromb Res 106(6): V285-294 (2002)) . However, some lower extremity vascular patients who underwent vascular surgery had a significantly lower long-term survival rate than patients with coronary heart disease who underwent surgery (J Am Coll Cardiol 51: 1588-1596 (2008). Most importantly, many patients with lower extremity ischemia because For various reasons, it is not suitable for vascular surgery or interventional surgery (Circulation Research (Circ Res) 105: 724-736 (2009)). Therefore, the use of angiogenesis factors and other therapeutic angiogenesis methods to promote angiogenesis and improve microcirculation, the more Increasingly recognized, especially in the treatment of lower extremity vascular diseases. Some angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and placenta-derived growth Factors (PDGF), etc., have been tested in lower limb ischemia animals and severe lower limb ischemia patients.

Tsurumi used VEGF for ischemic treatment for the first time, using the rabbit lower limb ischemia model, injecting VEGF plasmid into the ischemic site, thereby promoting angiogenesis and improving microcirculation (Circulation (Circulation) 94: 3281-3290 (1996) )).

Gene therapy is performed by introducing plasmid DNA into arterial vessels using a hydrogel-coated balloon catheter. Subsequently, clinical studies confirmed that intramuscular injection can also successfully express the transgene (Circulation (Circulation) 94: 3281-3290 (1996)). Baumgartner conducted preclinical trials by injecting VEGF ₁₆₅ plasmids into the limb muscles of patients with rest pain or intractable ulcers (Circulation 97: 1114-1123 (1998)). Folkman believes that this result needs to be confirmed by anatomical and physiological structures, including hemodynamics, angiography, pain relief, ischemic ulcer recovery, extremity recovery, and immunohistochemistry of endothelial proliferating cells. Validation (Circulation 97: 1108-1110 (1998)).

Makinen et al. compared the therapeutic effects of a plasmid encoding VEGF ₁₆₅ and an adenovirus encoding it on PAD exhibiting lameness and critical limb ischemia (CLI). Forty-five patients with PAD who participated in this percutaneous transluminal angioplasty were randomized to receive VEGF ₁₆₅ plasmid, VEGF _165- encoding adenovirus, and lactated Ringer's placebo (Mol Ther 6: 127–133 (2002) ). The treatment of VEGF ₁₆₅ has shown certain safety and significant angiogenesis effect, but in the clinical trials of VEGF ₁₆₅ , there is no significant difference between the treatment group and the control group.

Among the isoforms of VEGF, VEGF ₁₂₁ lacking the hairpin-binding domain is also thought to bind the protein complex. This is due to the relatively short half-life of regionally expressed VEGF ₁₂₁ in tissues. Therefore, it can only induce the early stages of angiogenesis, which means that its loose association with endothelial cells may lead to tissue edema. However, VEGF _165, which has a longer tissue half-life, can effectively mediate the entire period of angiogenesis.

The clinical trial of RAVE (Regional Angiogenesis with Vascular Endothelial Growth Factor) is the first randomized double-blind and placebo-controlled intramuscular injection of adenoviral vector for PAD treatment (Circulation (Circulation) 108: 1933-1938 (2003)) . Among them, 105 patients with claudication with unilateral motion limitation were randomly treated with intramuscular injection of low concentration AdVEGF ₁₂₁ , high concentration AdVEGF ₁₂₁ and placebo. After one quarter and six months of treatment, the researchers did not find a difference between the primary efficacy endpoint and the auxiliary efficacy endpoint among the three groups of patients, while the treatment of the AdVEGF ₁₂₁ group showed peripheral edema related to the dosage. This result is striking, as VEGF can increase the permeability of blood vessels, thus showing that AdVEGF ₁₂₁ is biologically active in this experiment.

Comerota et al. published the first clinical study of the FGF1 gene on CLI (J Vasc Surg 35:930-936 (2002)). The results showed that intramuscular injection of naked plasmid FGF1 (NV1FGF) in patients with peripheral arterial disease was effective in wound healing, pain relief and even improvement of percutaneous oxygen pressure. However, in the TALISMAN201 (Therapeutic Angiogenesis with Intramuscular NV1FGF Improves Amputation-Free Survivalin Patients with Critical Limb Ischemia) clinical trial, Nikol et al. conducted clinical phase II trials on patients with CLI (Molecular Therapy (Mol Ther) 16: 972–978 (2008)). These patients were randomized to receive intramuscular injections of NV1FGF (n=59) or placebo (n=66). After 25 weeks of treatment, 19.6% of patients in the treatment group and 14.3% of patients in the control group reached the primary efficacy endpoint. This result is not ideal, but the pooled cut-off rate in the experimental group was close to 37.3%, which is a significant reduction compared to 55.4% in the control group (hazard ratio = 0.498; P = 0.015). In addition, the mortality rate of patients treated with NV1FGF showed a downward trend. Both amputation and death are the main efficacy endpoints of clinical phase III trials of NV1FGF in patients with CLI.

Taniyama et al. studied exogenous HGF as a gene drug for the treatment of ischemic diseases, and achieved good results in lower limb ischemia models of rats and rabbits, which can promote angiogenesis, reduce the degree of ulceration, and relieve necrosis extent (Gene Ther 8:181-189 (2001)). Morishita and colleagues conducted a pilot study of HGF gene therapy and its safety assessment, which will provide the basis for the clinical phase II HGFSTAT study (hypertension (hypertension) 44: 203-209 (2004) ); Hypertension 33:1379-1384 (1999)). The 104 patients participating in this experiment will be randomized to receive placebo and three different doses of HGF plasmid injection (Circulation (Circulation) 118: 58-65 (2008)). Serious adverse events occurred in approximately 60% of the trials, but not in all groups. And it also proves that we don't need to worry about the safety of HGF plasmid therapy. However, when those patients who showed a TcPO2 increase of 15 mm Hg before treatment were excluded, the increase was most pronounced in patients who received the highest dose of HGF plasmid. This conclusion underscores how conventional results are often severely challenged when using surrogate endpoints to measure patients with severe PAD.

Another carrier of HGF has been applied in recent early clinical studies. VM202 includes the genomic cDNA hybrid of human HGF gene and HGF-X7, which can express various HGF isoforms through different splicing methods. In March 2009, the results of a treatment safety and dose gradient study (ClinicalTrials.govIdentifier: NCT00696124) covering 12 CLI patients provided preliminary evidence for the safety and biological activity of this treatment.

The transcription of HIF-1, as a typical gene master switch, can regulate multiple downstream targets in the physiological and pathological processes of angiogenesis. For example, strategies targeting HIF-1 or other regulators in oxygen-sensitive cells have been investigated for the treatment of ischemic diseases (Nat Med 9:677-684 (2003)) . And carried out experiments in the lower limb ischemia model of young and old mice, found that HIF-1 can promote angiogenesis, improve blood flow reperfusion, especially for old mice, this situation is more obvious (circulatory Research (Circ Res) 101: 1310-1318 (2007)). Rajagopalan et al conducted a safety assessment of a quantitative clinical trial of the modification and activation of HIF-1 in 38 patients with PAD and CLI. They did not find any negative effects of the approach (Circulation 115: 1234-1243 (2007)). At the same time, although the sample size they selected was relatively small, in these patients, the scholars observed the reduction of pain symptoms and the recovery of ulcers. In the WALK clinical study (ClinicalTrials.gov Identifier: NCT00117650), the researchers had a more randomized and more severe patient sample including 289 patients.

AGGF1, also known as VG5Q, is a congenital venous malformation hypertrophy syndrome (Klippel-Trenaunay Syndrome, KTS) discovered in 2004. KTS is a rare congenital vascular disease whose main symptoms are malformations and hyperplasia of capillaries, venous vessels, and lymphatic vessels. The disease was first classified, described, and defined by French doctors Klippel and Tranaunay in 1900. They have to endure physical pain such as the pain caused by the disease, and also have to bear the huge mental pressure caused by disability and deformity.

There are three main clinical features of KTS: skin capillary and lymphatic malformation and hyperplasia, often accompanied by red vascular spots (port-wine stains); premature varicose veins or venous malformations (Venous malformations or varicose veins); bone or Soft tissue overgrowth, usually manifested as unilateral extremity hypertrophy. Generally speaking, most patients have the above two characteristics, but the clinical diagnostic criteria of KTS are still these three basic characteristics.

Since KTS is a syndrome, it is often accompanied by many other clinical symptoms:

Capillary malformations (capillary malformations, CMs) are the most common skin vascular malformations. 95-100% of KTS patients have obvious capillary malformations and form vascular spots. The color varies from light to purple due to different symptoms. Red varies. Capillaries in KTS patients are relatively thin, and the endothelial cells lining them are generally flat.

Venous malformations (Venous malformations) will appear in 70-80% of KTS patients, but the manifestations of the symptoms are quite different, some are mild, with only small superficial vein abnormalities, and some are relatively dark, often appearing in large veins. area of lesions with ulceration. This symptom is less obvious in infancy, but with age, its symptoms gradually become more severe. Most varicose veins do not appear on the skin surface, but there are still many patients with KTS who have malformations in the internal veins of the affected legs, such as internal venous lumen varicose, missing venous valves, venous hypoplasia, and some deep veins are absent, and more Furthermore, malformations of the abdominal or pelvic veins have been found in some patients.

Lymphatic abnormalities (LMs) mostly refer to skin or subcutaneous lymphatic defects, which are more common in the face and neck of KTS patients. And about 20% of KTS patients have lymphatic fluid leaking from the lymphatic vessels.

In the family of KTS patients, Tian found a balanced translocation of t(5;11)(q3.3;p15.1) in the lymphocytes of a KTS patient by positional cloning. The precise translocation breakpoints of these two chromosomes, so as to clone the KTS candidate gene near the translocation breakpoints, AGGF1 (VG5Q) was cloned near the breakpoints of chromosome 5, and it is for this reason, this The gene was originally named VG5Q.

Subsequent studies found that the full-length protein of VG5Q has 718 amino acids. Through protein structure analysis, it was found that VG5Q contains multiple domains, including a loop structure at the N-terminus, and an OCTamer repeat domain (OCRE), Forkhead-related domains (FHA), with a G-patch domain at the C-terminus. According to the protein structure domain of CG5Q, people renamed VG5Q as AGGF1 ( Angio g enic Factor with G -patch and F HA domains 1 , AGGF1)

In 1995, Hofmann and Bucher discovered that the FHA domain ( Fork head- a ssociated domain) is a protein domain of a phosphokinase that can specifically recognize threonine. So far, more than 200 proteins contain this domain. This domain is involved in a series of reactions, such as signal transduction pathways, transcription, protein translation, DNA repair and protein degradation, etc. This domain is often involved in kinases, phosphatases, kinesins, transcription factors, RNA binding proteins, and some metabolic in the enzyme.

The protein sequence of AGGF1 is highly conserved among various species, especially the Forkhead-associated domain (FHA) and the C-terminal G-patch domain, but the functions of these domains of AGGF1 have not been studied in depth.

At present, in the prior art, there is no report about the ability of AGGF1 to promote angiogenesis.

Contents of the invention

The purpose of the present invention is to provide a new method for stimulating and promoting angiogenesis in ischemic tissue,

According to a first aspect of the present invention, these and other objects are achieved by providing a method of promoting revascularization of tissues in an organism comprising administering to the above-mentioned organism in need of such treatment an effective amount of the AGGF1 protein, or its AGGF1-bearing organism. Active fragments or analogs.

The AGGF1 may be human AGGF1, or AGGF1 of other mammals such as mice and rats. The amino acid sequence of human AGGF1 is shown in SEQ ID NO: 1 in the sequence listing; its coding nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence of AGGF1 in mice is shown in SEQ ID NO in the sequence listing Shown in: 3; Its coded nucleotide sequence is shown in SEQ ID NO: 4 in the sequence listing. As shown in the sequence table, the amino acid sequence of human AGGF1 has high homology with the amino acid sequence of mouse AGGF1. The AGGF1 genes in various species have a high degree of homology, and their amino acid sequences have a high degree of identity, so they can be used interchangeably in the present invention. In addition, variants, active fragments, conjugates and other derivatives of various AGGF1 genes can also be used.

According to another aspect of the present invention, the object of the present invention is achieved by providing a method for stimulating angiogenesis in ischemic tissue through angiogenesis, comprising administering an effective blood vessel stimulating amount of AGGF1 protein, or its AGGF1 biologically active protein Steps for fragments or analogues.

Post-infarct ischemic disease states are caused by decreased perfusion following decreased blood flow. This problem can be mitigated by reperfusion of the infarcted area with blood. Because most of the blood vessels and muscle cells in the infarcted area will die, reperfusion can only be achieved by increasing blood flow, which depends on the new blood vessels in the infarcted area.

Therefore, candidates for treatment of angiogenesis using AGGF1 include, among others, (1) patients with ischemic extremity or chronic venous leg ulcers; (2) patients with cerebral infarction; (3) patients with angiographically confirmed coronary artery disease. Patients with chronic stable angina (Carmeliet P and Collen D, Transgenic mouse models of angiogenesis and cardiovascular disease (review), Journal of Pathology, 2000; 190(3) 387-405); (4) patients with postnatal arterial dysmorphia; (5) Patients with restenosis after balloon angioplasty (Carmeliet P, Moons L, and Collen D, Mouse models of angiogenesis, arterial stenosis, atherosclerosis, and hemostasis (review), Cardiovascular Research, 1998; 39 (1 ) 8-33); (6) patients with ischemic cardiovascular disease; and (7) patients with end-stage coronary artery disease.

According to the present invention, there are a variety of techniques that can be used to administer AGGF1 that is not effective in promoting angiogenesis to patients suffering from ischemia or some other disease that can be alleviated by angiogenesis. The administered AGGF1 may be administered as naked DNA or as a carrier gene, or may be a recombinant protein, or may be a virus. Of course, regulatable vectors can be used if desired. These administration methods can also be administered together with some auxiliary materials, such as some polymer materials (resin, etc.), so as to improve the transfection efficiency and prolong the expression time. Direct intramuscular injection or intravenous injection can be used, electroporation can also be used, and it can also be attached to a stent for slow release, and ultrasonic microbubble technology or nanobubbles can also be used for drug delivery.

For example, naked plasmid DNA encoding AGGF1, or AGGF1-encapsulating virus, or AGGF1 protein can be administered to patients with myocardial ischemia by direct myocardial injection of AGGF1 during surgery or through microthoracotomy.

Also under the guidance of ultrasound, ultrasonic microbubbles can be split at the site of myocardial infarction or limb ischemia, thereby releasing AGGF1 to act on the target site. Mukherjee et al. used ultrasound-mediated contrast agent coated with human recombinant VEGF protein to significantly promote angiogenesis (J AmColl Cardiol 2000;35:1678–86). Enomoto et al. used ultrasonic microbubble technology to specifically release BMC cells to myocardial infarction sites in rats (Heart 2006;92:515–520).

Another possibility of AGGF1 administration is intramuscular injection of AGGF1 plasmid or AGGF1-encapsulated virus, mainly adeno-associated virus or retrovirus, etc. in the ischemic limb. Korpisalo et al. injected adenovirus directly into the muscle of ischemic lower limb rabbits (Circ. Res. 2008; 103; 1092-1099). Shimpo et al injected adeno-associated virus coated with VEGF directly into the leg muscles of rats with lower limb ischemia (Cardiovasc Res (2002) 53 (4): 993-1001 ). Hiraoka et al. directly injected the naked plasmid into the muscle of lower limb ischemic rats (Circulation 2003; 108; 2689-2696). Some people even use electroporation technology in order to increase the expression level. After injection of FGF plasmids into the hindlimb muscles of rabbits with lower limb ischemia, the expression of FGF was increased by electroporation (Journal of Surgical Research 120, 37–46 (2004)).

Recombinant AGGF1 protein can be injected intravenously to improve the degree of necrosis in ischemic sites and promote angiogenesis. Sumi et al injected recombinant high-density lipoprotein into lower limb ischemic mice through the tail vein to promote angiogenesis and improve adverse reactions caused by ischemia (ArteriosclerThromb Vasc Biol 2007;27;813-818).

In addition, the present invention also provides the use of AGGF1 protein or its active fragments in the preparation of drugs for promoting angiogenesis; wherein the AGGF1 protein can be natural AGGF1 protein or recombinant AGGF1 protein; and the drug can be an injection.

The present invention also provides the use of nucleotide fragments encoding AGGF1 protein or its active fragments in the preparation of drugs for promoting angiogenesis; wherein the nucleotide fragments can be connected with plasmids, or with adenovirus vectors and adeno-associated virus vectors or other replication defective retroviral vectors; the plasmid or virus containing AGGF1 is encapsulated in microvesicles.

The above drugs are used for the treatment of chronic stable angina pectoris due to coronary artery disease, critical limb ischemia, chronic venous leg ulcers, diabetic neuropathy, postnatal arterial insufficiency, restenosis, ischemic cardiovascular disease and A condition of end-stage coronary artery disease.

In another embodiment, the AGGF1 protein or its active fragment, or the nucleotide fragment encoding the AGGF1 protein or its active fragment and at least one selected from VEGF-A, VEGF-B, VEGF-C, PIGF, PDGF Together the growth factors of -A, PDGF-B, PDGF-C, HGF and FGF are used to promote angiogenesis.

In another embodiment, the AGGF1 protein or its active fragment, or the nucleotide fragment encoding the AGGF1 protein or its active fragment is used together with cells such as endothelial progenitor cells, bone marrow stem cells and/or cardiac stem cells to promote the formation of blood vessels .

Beneficial effects of the present invention: Introducing exogenous therapeutic AGGF1 to ischemic tissue or parts, it is found that the blood flow rate in the ischemic part can be significantly increased, angiogenesis can be promoted, oxygen supply in the hypoxic part can be improved, and ischemic symptoms can be alleviated. Improve exercise capacity and significantly reduce the degree of necrosis.

Description of drawings

The present invention can be described in further detail by reference to the following experiments illustrating the activity of AGGF1 in promoting angiogenesis in ischemic tissue, the accompanying drawings showing the results of these experiments, wherein:

Fig. 1 is a diagram of the blood flow of the femoral artery of mice before operation. The blood flow of the femoral artery of normal mice was detected by small animal high-frequency ultrasound Doppler in the mode of color pulse Doppler.

Fig. 2 is a graph showing the blood flow of femoral artery of mice after operation. The blood flow of the femoral artery of mice with ischemic lower extremity was detected by small animal high-frequency ultrasound Doppler in the mode of color pulsed Doppler. Through the frequency spectrum, it was found that the proximal end of the femoral artery was ligated or narrowed to a greater extent, the blood supply to the lower extremities was obviously insufficient, and the maximum blood flow velocity during systole decreased significantly. If the frequency spectrum does not change, and the maximum systolic blood flow velocity does not decrease to a large extent, it is considered that the operation is unsuccessful, and the mice that fail to make models should be excluded.

Fig. 3 is a graph showing the ratio of the blood flow velocity of the left and right legs detected by ultrasound at different time points. Because there is a certain correlation between blood flow and temperature, in order to exclude the influence of these factors, the statistical method of blood flow ratio is used.

Fig. 4 is a diagram of mouse tissue necrosis at different time points. 0, no necrosis; 1, one toe necrosis; 2, two or more toes necrosis; 3, foot necrosis; 4, whole leg necrosis; 5, spontaneous amputation. All groups were n=9.

Fig. 5 is a score chart of mouse hindlimb behavior ability at different time points. 1. For the pulling of the tail, the hind limbs can stretch, but the toes do not respond; 2. Neither the hind limbs nor the toes respond; 3. The hind limbs drag. All groups were n=9.

Fig. 6 is a diagram of immunohistochemical results of mouse gastrocnemius muscle administered for 7 days, 14 days and 28 days. The presence of neovascularization marked by the vessel-specific marker CD31.

Fig. 7 is a graph showing the statistical results of new blood vessels in gastrocnemius muscle of mice administered for 28 days. For the statistics of new blood vessels marked by the blood vessel-specific marker CD31, FIG. 7A is a graph of the number of new blood vessels per unit area. Figure 7B is a graph of the number of neovascularization relative to muscle fascicles.

Fig. 8 is a schematic diagram of HE staining of mouse gastrocnemius muscle tissue sections. Fig. 8C is an enlarged view of the box in the picture of Fig. 8A; Fig. 8D is an enlarged view of the box in the picture of Fig. 8C.

Detailed ways

Materials and Experimental Methods

1) Unilateral lower extremity artery ligation

Male C57BL/6 mice at 10-12 weeks were anesthetized with pentobarbital sodium (50 mg/kg) intraperitoneally, and allowed to breathe spontaneously during the operation. Depilate the left and right sides of the mouse from the groin to the thigh with depilatory cream to make the surface clean. Cut the skin transversely and bluntly separate the blood vessels and nerves. Carefully separate the vein from the artery. The iliac artery was ligated on the right side with 6-0 silk suture. The vessel was dissected down the iliac artery, and the femoral artery was subsequently ligated. During ligation, do not injure the vein and accompanying nerve. The artery on the left side was wrapped around a circle with 6-0 silk thread, which was used as a control in the experiment, that is, the sham-operated leg.

2) Plasmid preparation of AGGF1 and bFGF

The method for preparing AGGF1 and bFGF plasmids has been previously described using a recombinant expression vector that has integrated the target gene into a commonly used gene expression vector. AGGF1 and bFGF plasmids were prepared according to the method in Circulation 108:2689-2696 (2003). . AGGF1 and bFGF plasmids were purified free of endotoxin by phenol chloroform extraction and potassium acetate precipitation.

3) Ultrasonic detection of femoral artery blood flow in small animals

Blood flow in mice was detected using a Vevo 2100 high-resolution small animal ultrasound system (Visualsonics Inc, Toronto, Canada). The mouse was anesthetized with 1% isopentane, placed on the mouse board at 37°C, and the hair from the thigh to the calf of the two legs of the mouse was removed with depilatory cream to reduce the effect of the hair on the ultrasonic detection. Influence. The MS550 type probe was used for detection. The blood velocity of the femoral artery was detected in the color pulsed Doppler mode. Before the operation, the blood flow of the femoral artery of the mouse was detected as shown in Figure 1, and on the first day after the operation, the ultrasonic detection was performed again, as shown in Figure 2, to determine whether the lower limb ischemia model was successful. The criterion for the success of the operation is that the pulse Doppler spectrum is abnormal, basically there is no maximum systolic blood flow, and the blood flow velocity is significantly reduced, which is basically less than 10% of the pre-operation.

4) Detection of tissue necrosis and behavior

Humans who did not participate in the experiment scored the mice for tissue necrosis and performance. The scoring criteria for tissue necrosis (Eur J Pharmacol 634(1-3): 121-131 (2010)) are: 0, no necrosis; 1, one toe necrosis; 2, two or more toes necrosis; 3, foot necrosis ; 4, necrosis of the whole leg; 5, self-amputation, as shown in Figure 4. Through scoring, we found that compared with the empty plasmid, AGGF1 can improve the necrosis of ischemic tissue, alleviate the ischemic symptoms of mice, and thus alleviate the pain caused by ischemia.

The scoring standard of behavioral ability (Circle Research (Circ Res) 101: 1310-1318 (2007)) is: 0, the hind limbs respond normally to the pulling of the tail; No response; 2, no response from the hind limbs and toes; 3, dragging of the hind limbs, as shown in Figure 5. Under the action of AGGF1, the mobility of the hindlimbs of mice with lower limb ischemia was significantly enhanced, which was beneficial to alleviate the ischemic symptoms of mice. The enhancement of exercise capacity is also conducive to angiogenesis in the ischemic lower limbs, thereby improving various adverse consequences caused by ischemia (Circulation (Circulation) 122: 707-716 (2010)).

5) Immunohistochemical detection

Twenty-nine days after the operation, the mice were sacrificed, the gastrocnemius muscle was isolated, fixed overnight in 4% paraformaldehyde, embedded in paraffin, and sectioned. CD31, a specific marker for neovascularization, was used for immunohistochemical detection, as shown in FIG. 6 .

Example 1

Targeting ischemic tissue, using the AGGF1 gene for angiogenesis therapy

According to the method described above, a mouse model of lower limb ischemia was established.

The method of intramuscular injection of DNA plasmid was used to treat the ischemic part of the mouse lower limb ischemia model for angiogenesis. One day after the operation, a single point injection of 0.15 ml of the plasmid was performed below the ligation site, ie, the gastrocnemius muscle site.

Blood flow detection was performed at 8 days, 15 days, and 29 days after surgery, as shown in Figure 3. Eight days after surgery, the blood flow velocity ratio of the left and right legs of the AGGF1-treated mice was 0.22 ± 0.10, while that of the mice in the control group was 0.10 ± 0.04, (P < 0.05, n=9). At 29 days after surgery, the blood flow velocity ratio of the left and right legs of the AGGF1-treated mice was 0.31±0.06, while that of the mice in the control group was 0.14±0.03, (P ＜ 0.01, n=9) . It is found that AGGF1 treatment can indeed significantly increase the blood flow velocity and enhance the blood flow reperfusion ability.

According to the results of immunohistochemistry, it was found that exogenous AGGF1 could significantly stimulate the formation of microvessels. After AGGF1 stimulation for 28 days, the number of new blood vessels reached 0.934±0.413/mm ² , while that of the control group was only 0.538±0.388/mm ² (P < 0.01), as shown in Figure 7A. At the same time, the number of new blood vessels was 439.2±250.1, and 172.5±109.4 in the control group (P<0.05), as shown in Figure 7B.

By HE staining of the sections, it was found that the concentration of nuclei was not significant in mice injected with AGGF1. Moreover, the number of necrotic muscle fibers also decreased significantly, as shown in Figure 8. Thus, AGGF1 can significantly promote angiogenesis.

Example 2:

For ischemic tissue, using recombinant AGGF1 protein for angiogenesis treatment

According to the method described above, a mouse model of lower limb ischemia was established.

After the mouse lower limb ischemia model was established, the purified AGGF1 protein was injected through the tail vein on the second day after the operation to induce angiogenesis in the ischemic tissue and alleviate the adverse reactions caused by ischemia. Tissue necrosis and behavioral ability were detected according to the above method, and it was found that the hindlimb motor ability of mice given AGGF1 protein was significantly enhanced, and the degree of necrosis was also improved.

Example 3:

Targeting ischemic tissue, angiogenesis therapy using AGGF1 adeno-associated virus

The cDNA encoding AGGF1 is cloned into the adeno-associated virus vector, transfected, purified, identified, and its titer is determined to obtain the AGGF1 adeno-associated virus.

According to the method described above, a mouse model of lower limb ischemia was established. On the second day of surgery, the purified AGGF1 adeno-associated virus was injected through the tail vein. Femoral artery blood flow, tissue necrosis and behavioral ability were detected at different time points after injection. The test results showed that, compared with the control (empty adeno-associated virus vector lacking the cDNA encoding AGGF1), the femoral artery blood flow of mice injected with AGGF1 adeno-associated virus was significantly increased, the hindlimb motor ability of the mice was significantly enhanced, and the degree of necrosis was also reduced. improved. These experimental results confirmed that AGGF1 AAV can effectively induce angiogenesis in ischemic tissue and alleviate the adverse reactions caused by ischemia.

In the above three examples, the plasmids encoding AGGF1, the AGGF1 protein, and the AGGF1 adeno-associated virus were injected into the mouse lower limb ischemia model. adverse reactions. Thus confirming that AGGF1 can induce angiogenesis and treat diseases and other adverse reactions caused by local ischemia.

Although the present invention has been described in detail through the above examples, it should be understood that these examples are only for the purpose of description, and should not be construed as limiting the present invention.

Claims (12)

1.AGGF1 albumen or its active fragment promote the purposes in the angiopoietic medicine in preparation.

2. according to the purposes of claim 1, wherein said AGGF1 albumen is reorganization AGGF1 albumen.

3. according to the purposes of claim 1 or 2, wherein said medicine is an injection.

4. the nucleotide fragments of coding AGGF1 albumen or its active fragment promotes the purposes in the vascularization medicine in preparation.

5. according to the purposes of claim 4, the nucleotide fragments of wherein said coding AGGF1 albumen or its active fragment effectively is connected with plasmid.

6. according to the purposes of claim 4, the nucleotide fragments of wherein said coding AGGF1 albumen or its active fragment effectively is connected with the retroviral vector of adenovirus vector, gland relevant viral vector or other replication defectives.

7. according to the purposes of claim 5 or 6, plasmid or the virus packets of the wherein said AGGF1 of containing are rolled in the microvesicle.

8. according to the purposes of claim 1 or 4, wherein said medicine is used to treat the ischemic disease.

9. the method for claim 8, wherein said disease be selected from chronic stable angina pectoris that coronary artery disease causes, critical limb ischemia, chronic venous property leg ulcer, diabetic neuropathy, birth artery functional defect, restenosis, ischemic cardiovascular and whole latter stage coronary artery pathological changes disease.

10. according to the purposes of claim 1 or 4, the nucleotide fragments of wherein said AGGF1 albumen or its active fragment or coding AGGF1 albumen or its active fragment is used to promote vascularization with the somatomedin of at least a VEGF-A of being selected from, VEGF-B, VEGF-C, PIGF, PDGF-A, PDGF-B, PDGF-C, HGF and FGF.

11. according to the purposes of claim 1 or 4, the nucleotide fragments of wherein said AGGF1 albumen or its active fragment or coding AGGF1 albumen or its active fragment is used to promote the formation of blood vessel with cells such as endothelial progenitor cells, bone marrow stem cell and/or myocardium stem cell.

12. the angiopoietic method of the promotion of non-therapeutic purposes comprises the nucleotide fragments of the AGGF1 albumen that gives the vascular stimulation effective dose or its active fragment and/or coding AGGF1 albumen or its active fragment.

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