Angiogenesis In Ischemic Tissues Research Articles

The Inhibition of γ-Aminobutyric Acid B1 Receptor Regulates Angiogenesis via the Hippo/YAP Signaling Pathway

Promoting the establishment of collateral circulation is essential for chronic lower extremity ischemia. However, no effective therapeutic drugs have yet been developed. Recent studies discovered that in the peripheral arteries, there are GABAB1 receptors expressed in endothelial cells and smooth muscle cells, these receptors may have some effects in regulating vascular functions, but the precise mechanism is not yet clear. This study explores the effect of GABAB1 receptor inhibition on angiogenesis and its regulatory mechanism. The expression of GABAB1 in HUVECs was knocked down using shRNA transfection, and effects in HUVECs' proliferation, migration, and tube formation ability were detected. Western blot and RT-PCR were used to verify the signal pathway. The murine hind limb ischemia model was used to verify the effect of CGP35348, an antagonist of GABAB1R, on the recovery of blood flow perfusion and angiogenesis in ischemic tissues. Cell proliferation, migration, and tube formation ability were improved after GABAB1 receptor knockdown in HUVECs. The phosphorylation of the HIPPO/YAP pathway decreased, while the effect of promoting angiogenesis increased. After treating the ischemic hindlimbs of mice with GABAB1 receptor antagonists, the blood flow perfusion recovered, and the angiogenesis increased. These findings demonstrate the effect of GABAB1 receptor inhibition on the HIPPO/YAP pathway in regulating angiogenesis, suggesting that inhibiting GABAB1 receptor levels might be a novel approach for chronic lower extremity ischemia diseases.

Stem cell factor and cKIT modulate endothelial glycolysis in hypoxia.

In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.

Counteracting Age-Related Netrin-1 Signaling Insufficiency Ameliorates Endothelial Cell Senescence and Angiogenesis Impairment.

Senescent cells that accumulate are regarded as promising therapeutic targets. However, senolytic therapy failed to achieve satisfactory results. We previously discovered that young human plasma improved vascular endothelial cell senescence, and UNC5B might be a novel intervention target. Netrin-1, as a natural ligand of UNC5B, plays roles in multiple age-related vascular disorders, but its involvement in aging is still unclear. Here, we observed a significant decrease in plasma Netrin-1 levels in old healthy subjects compared to the young. In vivo, adeno-associated-virus-mediated delivery of Netrin-1 into aged mice significantly improved functional recovery in a model of hindlimb ischemia, promoted angiogenesis in ischemic tissues, and activated the endothelial nitric oxide synthase. Furthermore, we revealed that low-dose Netrin-1 recombinant protein significantly reduced senescence-associated-β-galactosidase-positive cells, inhibited the P53 pathway, promoted cell migration, increased tubule formation, and elevated nitric oxide production in senescent endothelial cells. However, UNC5B inhibition blocked the pro-angiogenesis effect of low-dose Netrin-1 on senescent cells or aortic rings. In summary, this study depicts that modulating Netrin-1 signaling can result in improved vascular health and Netrin-1 may have therapeutic potential for age-related ischemic diseases.

Vasostatin-2 associates with coronary collateral vessel formation in diabetic patients and promotes angiogenesis via angiotensin-converting enzyme 2.

Members of the chromogranin family play a role in angiogenesis. One such biologically active peptide, generated through the processing of chromogranin A, is vasostatin-2. This study aimed at assessing the association of serum vasostatin-2 levels with coronary collateral vessels (CCV) in diabetic patients with chronic total occlusions (CTO) and the effects of vasostatin-2 on angiogenesis in diabetic mice with hindlimb or myocardial ischemia. Serum levels of vasostatin-2 in 452 diabetic CTO patients were evaluated. The status of CCV was categorized according to the Rentrop score. Vasostatin-2 recombinant protein or phosphate-buffered saline were then injected intraperitoneally in diabetic mouse models of hindlimb or myocardial ischemia, followed by laser Doppler imaging and molecular biology examinations. The effects of vasostatin-2 were also ascertained in endothelial cells and macrophages, with mechanisms clarified using ribonucleic acid (RNA) sequencing. Serum levels of vasostatin-2 were significantly different and progressively higher across Rentrop score 0, 1, 2, and 3 groups (P < .001), with significantly lower levels in patients with poor CCV (Rentrop score 0 and 1) than in those with good CCV (Rentrop score 2 and 3) (P < .05). Vasostatin-2 significantly promoted angiogenesis in diabetic mice with hindlimb or myocardial ischemia. RNA-seq analyzes verified an angiotensin-converting enzyme 2 (ACE2)-mediated vasostatin-2-induction of angiogenesis in ischemic tissues. Lower serum levels of vasostatin-2 are associated with poor CCV in diabetic CTO patients compared with patients with good CCV. Vasostatin-2 significantly promotes angiogenesis in diabetic mice with hindlimb or myocardial ischemia. Such effects are mediated by ACE2.

The RGD-modified self-assembling D-form peptide hydrogel enhances the therapeutic effects of mesenchymal stem cells (MSC) for hindlimb ischemia by promoting angiogenesis

Mesenchymal stem cell (MSC)-based therapy was an ideal strategy for therapeutic angiogenesis in ischemic tissues. However, limitations such as heterogeneous proangiogenic nature and low cell survival following transplantation is a key challenge for clinical applications. Biofunctional materials such as extracellular matrix (ECM) and hydrogels are considered with great promise for improved therapeutics by constructing a supportable 3D microenvironment niche for transplanted cells. Here, we employ the RGD (Arg-Gly-Asp)-modified self-assembling D-form peptide hydrogel (Nap-DFDFKGRGD) to serve as the co-transplantation platform for human placental MSCs (hP-MSCs). Our data showed that this D-form hydrogel possessed excellent biocompatibility and biostability, exerting an anti-apoptosis capacity and improved cell viability in vitro. Furthermore, we found that the self-assembled hydrogel improved cell survival, as well as promoted collateral vessel revascularization by bioluminescence imaging (BLI) and micro-CT imaging analysis. Further study confirmed significant up-regulation of proangiogenic cytokines of hP-MSCs in vivo. These results demonstrated the improvements of therapeutic efficacy of hP-MSCs in terms of tissue regeneration and functional recovery in the murine hindlimb ischemia (HLI) model. In conclusion, the strategy provided a promising option for ischemia diseases by improving the proangiogenic effects of MSCs and further for enhancing the therapeutic efficiency of MSCs with the RGD motif and the D-form peptide hydrogel.

MiR-548j-5p regulates angiogenesis in peripheral artery disease

Peripheral artery disease (PAD) is a vascular disease involving diffuse atherosclerosis, and is associated with increased cardiovascular mortality and morbidity. Critical limb ischemia (CLI) is the most severe complication of PAD. In addition to medical and interventional treatment, therapeutic angiogenesis is a novel therapy for PAD. Circulating microRNAs (miRNAs) are considered key regulators of gene expression, but their role in ischemic-induced angiogenesis is poorly-characterized. There is currently a limited understanding of the specific miRNAs associated with PAD. To determine the regulation of miRNAs, we obtained miRNA profiles using RNA isolated from patients with PAD and a control group. The effects of specific miRNAs on angiogenesis were evaluated by assessing the in vitro angiogenic function of endothelial progenitor cells (EPCs), performing an in vivo angiogenesis assay, and employing a mouse hindlimb ischemic model. Our results demonstrated that circulating miR-548j-5p was significantly reduced in patients with PAD as compared with the controls. miR-548j-5p promoted EPC angiogenesis by enhancing migration and tube formation. The endothelial nitric oxide synthase (NOS) and stromal cell-derived factor (SDF)-1 signaling pathways appeared to be potential targets of miR-548j-5p. Furthermore, the results of a directed in vivo angiogenesis assay of EPCs and a hindlimb ischemia mouse model demonstrated that miR-548j-5p enhanced the capillary density and blood flow recovery in hindlimb ischemia. In conclusion, our data indicated that up-regulation of miR-548j-5p promotes angiogenesis in ischemic tissue and may represent a novel therapeutic approach for PAD.

A functional extracellular matrix biomaterial enriched with VEGFA and bFGF as vehicle of human umbilical cord mesenchymal stem cells in skin wound healing

The construction of microvascular network is one of the greatest challenges for tissue engineering and cell therapy. Endothelial cells are essential for the construction of network of blood vessels. However, their application meets challenges in clinic due to the limited resource of autologous endothelium. Mesenchymal stem cells can effectively promote the angiogenesis in ischemic tissues for their abilities of endothelial differentiation and paracrine, and abundant sources. Extracellular matrix (ECM) has been widely used as an ideal biomaterial to mimic cellular microenvironment for tissue engineering due to its merits of neutrality, good biocompatibility, degradability, and controllability. In this study, a functional cell derived ECM biomaterial enriched with VEGFA and bFGF by expressing the collagen-binding domain fused factor genes in host cells was prepared. This material could induce endothelial differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) and promote angiogenesis, which may improve the healing effect of skin injury. Our research not only provides a functional ECM material to inducing angiogenesis by inducing endothelial differentiation of hUCMSCs, but also shed light on the ubiquitous approaches to endow ECM materials different functions by enriching different factors. This study will benefit tissue engineering and regenerative medicine researches.

Personalized Cell Therapy for Patients with Peripheral Arterial Diseases in the Context of Genetic Alterations: Artificial Intelligence-Based Responder and Non-Responder Prediction.

Stem/progenitor cell transplantation is a potential novel therapeutic strategy to induce angiogenesis in ischemic tissue, which can prevent major amputation in patients with advanced peripheral artery disease (PAD). Thus, clinicians can use cell therapies worldwide to treat PAD. However, some cell therapy studies did not report beneficial outcomes. Clinical researchers have suggested that classical risk factors and comorbidities may adversely affect the efficacy of cell therapy. Some studies have indicated that the response to stem cell therapy varies among patients, even in those harboring limited risk factors. This suggests the role of undetermined risk factors, including genetic alterations, somatic mutations, and clonal hematopoiesis. Personalized stem cell-based therapy can be developed by analyzing individual risk factors. These approaches must consider several clinical biomarkers and perform studies (such as genome-wide association studies (GWAS)) on disease-related genetic traits and integrate the findings with those of transcriptome-wide association studies (TWAS) and whole-genome sequencing in PAD. Additional unbiased analyses with state-of-the-art computational methods, such as machine learning-based patient stratification, are suited for predictions in clinical investigations. The integration of these complex approaches into a unified analysis procedure for the identification of responders and non-responders before stem cell therapy, which can decrease treatment expenditure, is a major challenge for increasing the efficacy of therapies.

A facile approach to therapeutic angiogenesis using a platelet concentrate and microsphere composite

AbstractStimulating revascularization of under perfused tissue is a key challenge in regenerative medicine. A variety of approaches using soluble biotherapeutic agents have been investigated in recent years but each have yielded limited potency or raised concerns regarding safety. This study reports on the finding that the pro‐angiogenic secretome released from platelet concentrates is significantly enhanced when combined with highly porous biodegradable polymer microspheres. These findings suggest a topically applied autologous platelet concentrate‐biomaterials based approach may serve as a facile and broadly applicable platform to achieve therapeutic angiogenesis in ischemic tissue or where preservation of tissue perfusion and viability is required.

Important Role of Concomitant Lymphangiogenesis for Reparative Angiogenesis in Hindlimb Ischemia.

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Gangrene, revascularization, and limb function improved with E-selectin/adeno-associated virus gene therapy

ObjectiveNovel therapeutic angiogenic concepts for critical limb ischemia are still needed for limb salvage. E-selectin, a cell-adhesion molecule, is vital for recruitment of the stem/progenitor cells necessary for neovascularization in ischemic tissues. We hypothesized that priming ischemic limb tissue with E-selectin/adeno-associated virus (AAV) gene therapy, in a murine hindlimb ischemia and gangrene model, would increase therapeutic angiogenesis and improve gangrene. MethodsFVB/NJ mice were given intramuscular hindlimb injections of either E-selectin/AAV or LacZ/AAV and then underwent induction of gangrene via femoral artery ligation and concomitant systemic injections of the nitric oxide synthesis inhibitor L-NAME (L-NG-Nitro arginine methyl ester; 40 mg/kg). Gangrene was evaluated via the Faber hindlimb appearance score. The rate of ischemic limb reperfusion and ischemic tissue angiogenesis were evaluated using laser Doppler perfusion imaging and DiI perfusion with confocal laser scanning microscopy of the ischemic footpads, respectively. The treadmill exhaustion test was performed on postoperative day (POD) 8 to determine hindlimb functionality. ResultsThe E-selectin/AAV–treated mice (n = 10) had decreased Faber ischemia scores compared with those of the LacZ/AAV–treated mice (n = 7) at both PODs 7 and 14 (P < .05 and P < .01, respectively), improved laser Doppler perfusion imaging reperfusion indexes by POD 14 (P < .01), and greater gangrene footpad capillary density (P < .001). E-selectin/AAV–treated mice also had improved exercise tolerance (P < .05) and lower relative muscular atrophy (P < .01). ConclusionWe surmised that E-selectin/AAV gene therapy would significantly promote hindlimb angiogenesis, reperfusion, and limb functionality in mice with hindlimb ischemia and gangrene. Our findings highlight the reported novel gene therapy approach to critical limb ischemia as a potential therapeutic option for future clinical studies.

Biodegradable Films: Microporous Biodegradable Films Promote Therapeutic Angiogenesis (Adv. Healthcare Mater. 17/2020)

In article number 2000806 by Richard M. Day and co-workers, highly porous biodegradable films are prepared using thermally-induced phase separation. In vivo implantation of the films into ischemic tissue stimulates increased expression of pro-angiogenic growth factor genes. This provides a favourable local environment for neovascularization, increased tissue perfusion, and therapeutic angiogenesis in ischemic tissue. The findings pave the way to establishing new materials-based strategies to achieve therapeutic angiogenesis.

Notoginsenoside R1 activates the Ang2/Tie2 pathway to promote angiogenesis

BackgroundTherapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce. PurposeWe aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway. MethodsWe examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). The aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in the zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis. ResultsWe found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. NR1 also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1. ConclusionsOur results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.

A Study of novel mechanism of hypoxic preconditioned CD34+cells improving therapeutic efficacy for critical limb ischemia: role of exosomal miRNA-378a-5p

Background & Aim CD34+cells transplantation has shown therapeutic angiogenesis efficacy in treating critical limb ischemia. Hypoxic preconditioning can increase the proangiogenic ability. As the main component of the paracrine secretion of CD34+ cells, CD34+ cell exosome (CD34-Exo) plays an important role. In this research, we aim to investigate the proangiogenic mechanism of hypoxic preconditioned CD34 exosome (HCC34-Exo) at the level of exsosomal miRNA. Methods, Results & Conclusion RNAseq screen showed 3.40±2.85 (means±standrad deviations) folds (n=6) of mi-RNA-387a-5p in the HCC34-Exo than in the normoxic preconditioned CD34+ cell exosomes (NCC34-Exo), and this was validated by qRT-PCR. In vitro experiments such as transwell co-culture model, exosome secretion-blocked experiment, exosome tracking experiment demonstrated that miRNA-378a-5p was directly transferred from the CD34+ cells through exosomes of CD34+ cells to human umbilical vein endothelial cells (HUVECs). Tube formation experiment demonstrated the enhancement of the HUVECs function, significant increase was observed concerning branching points in HCC34-Exo group, which equals to 2.15±0.46 folds of that in blank control group, 1.80±0.62 folds of that in anti-miRNA-378 HCC34-Exo group and 1.32±0.34 folds of that in NCC-Exo group (all p Our results have showed that HCC34 might promote therapeutic angiogenesis in ischemic tissue through exosomal miR-378-5p. Identification of the mechanism will provide important theoretical evidence for the therapeutic efficacy of HCC34 and its exsosomal miRNA in treating critical limb ischemia and may offer a new idea for potential treatment strategies in the future.

Next‐gen mRNA‐enhanced Cell Therapy

A new therapeutic field is emerging as obstacles are overcome to the clinical applications of mRNA drugs. The immunogenicity of mRNA has been reduced by the use of modified nucleosides and ultra‐purification. The stability and translatability of mRNA is increased by attention to optimizing codon use, the translational ramp, the 3′ and 5′UTRs, and by adding nuclease‐resistant secondary structure. The delivery of mRNA depends upon the application, but advances in targeted nanoparticle delivery vehicles, electro‐ or mechanoporation, microneedles and scaffolds have increased the delivery of therapeutic mRNA to the desired tissue. Because mRNA is non‐integrating, it is safer for human use than DNA‐based gene therapies.Cell therapies, including stem cell therapies for regenerative medicine, may be enhanced by mRNA‐based strategies. For example, in immunodeficient mice bearing a human B cell lymphoma, chimeric antigen receptor T (CART) cell therapy against the tumor was improved by treating the CART cells with human telomerase mRNA (hTERT mRNA). Treatment with hTERT mRNA increased telomere length of CART cells; reduced senescence of the cells with expansion; increased therapeutic cell product; and increased the anti‐tumor effect in vivo. Similarly, the ability of mesenchymal stem cells (MSCs) to induce angiogenesis in ischemic tissue was enhanced by increasing MSC telomerase activity and telomere length.Induced pluripotent stem cells (iPSCs) can be generated using mRNA encoding the Yamanaka factors, avoiding the viral integration of DNA encoding these factors. The lack of integration and its potential off‐target effects may be beneficial for iPSC‐derived cells for regenerative applications. Furthermore, mRNA encoding lineage‐determination factors can facilitate directed differentiation of these iPSCs to the therapeutic cells of choice. Alternatively, mRNA encoding the differentiation factors of choice can be administered directly to the tissue in scaffolds or nanocarriers to enhance endogenous cellular regenerative processes.Because mRNA constructs encoding any protein of interest can be generated rapidly in a cGMP fashion, and because many effective delivery vehicles are also now cGMP capable, mRNA therapeutics is a fast, flexible, and disruptive technology that has great promise in the field of regenerative medicine and cell therapy.

Impact of Therapeutic Angiogenesis Using Autologous Bone Marrow-derived Mononuclear Cell Implantation in Patients with No-option Critical Limb Ischemia.

Recently, the limb salvage rate of patients with critical limb ischemia (CLI) has been improved due to the development of revascularization and wound care treatment. However, many patients with CLI are refractory to standard treatments, including revascularization such as endovascular treatment or surgical bypass. Establishment of a new cell therapy is required to improve the limb salvage rate and prognosis in patients with CLI. In 1997, endothelial progenitor cells were found to be derived from the bone marrow to circulate as CD34 surface antigen positive cells in peripheral blood and to affect therapeutic angiogenesis in ischemic tissues. Later, therapeutic angiogenesis using autologous bone marrow-derived mononuclear cell (BM-MNC) implantation was performed for patients with no-option CLI in clinical practice. Several reports showed the safety and efficacy of the BM-MNC implantation in patients with CLI caused by arteriosclerosis obliterans, thromboangiitis obliterans (TAO), and collagen diseases. In particular, in patients with CLI caused by TAO, limb salvage rate was significantly improved compared with standard treatments. The BM-MNC implantation may be feasible and safe in patients with no-option CLI. Here, we review the efficacy of BM-MNC implantation in no-option CLI, with a focus on therapeutic angiogenesis.

A histone deacetylase 7-derived peptide promotes vascular regeneration via facilitating 14-3-3γ phosphorylation.

Histone deacetylase 7 (HDAC7) plays a pivotal role in the maintenance of the endothelium integrity. In this study, we demonstrated that the intron‐containing Hdac7 mRNA existed in the cytosol and that ribosomes bound to a short open reading frame (sORF) within the 5′‐terminal noncoding area of this Hdac7 mRNA in response to vascular endothelial growth factor (VEGF) stimulation in the isolated stem cell antigen‐1 positive (Sca1+) vascular progenitor cells (VPCs). A 7‐amino acid (7A) peptide has been demonstrated to be translated from the sORF in Sca1+‐VPCs in vitro and in vivo. The 7A peptide was shown to receive phosphate group from the activated mitogen‐activated protein kinase MEKK1 and transfer it to 14‐3‐3 gamma protein, forming an MEKK1‐7A‐14‐3‐3γ signal pathway downstream VEGF. The exogenous synthetic 7A peptide could increase Sca1+‐VPCs cell migration, re‐endothelialization in the femoral artery injury, and angiogenesis in hind limb ischemia. A Hd7‐7sFLAG transgenic mice line was generated as the loss‐of‐function model, in which the 7A peptide was replaced by a FLAG‐tagged scrabbled peptide. Loss of the endogenous 7A impaired Sca1+‐VPCs cell migration, re‐endothelialization of the injured femoral artery, and angiogenesis in ischemic tissues, which could be partially rescued by the addition of the exogenous 7A/7Ap peptide. This study provides evidence that sORFs can be alternatively translated and the derived peptides may play an important role in physiological processes including vascular remodeling.

Local pharmacological induction of angiogenesis: Drugs for cells and cells as drugs

The past decades have seen significant advances in pro-angiogenic strategies based on delivery of molecules and cells for conditions such as coronary artery disease, critical limb ischemia and stroke. Currently, three major strategies are evolving. Firstly, various pharmacological agents (growth factors, interleukins, small molecules, DNA/RNA) are locally applied at the ischemic region. Secondly, preparations of living cells with considerable bandwidth of tissue origin, differentiation state and preconditioning are delivered locally, rarely systemically. Thirdly, based on the notion, that cellular effects can be attributed mostly to factors secreted in situ, the cellular secretome (conditioned media, exosomes) has come into the spotlight. We review these three strategies to achieve (neo)angiogenesis in ischemic tissue with focus on the angiogenic mechanisms they tackle, such as transcription cascades, specific signalling steps and cellular gases. We also include cancer-therapy relevant lymphangiogenesis, and shall seek to explain why there are often conflicting data between in vitro and in vivo. The lion's share of data encompassing all three approaches comes from experimental animal work and we shall highlight common technical obstacles in the delivery of therapeutic molecules, cells, and secretome. This plethora of preclinical data contrasts with a dearth of clinical studies. A lack of adequate delivery vehicles and standardised assessment of clinical outcomes might play a role here, as well as regulatory, IP, and manufacturing constraints of candidate compounds; in addition, completed clinical trials have yet to reveal a successful and efficacious strategy. As the biology of angiogenesis is understood well enough for clinical purposes, it will be a matter of time to achieve success for well-stratified patients, and most probably with a combination of compounds.

Enrichment of vascular endothelial growth factor secreting mesenchymal stromal cells enhances therapeutic angiogenesis in a mouse model of hind limb ischemia

Critical limb ischemia, a severe manifestation of peripheral artery disease, is emerging as a major concern in aging societies worldwide. Notably, cell-based gene therapy to induce angiogenesis in ischemic tissue has been investigated as treatment. Despite many studies demonstrating the efficacy of this approach, better therapies are required to prevent serious sequelae such as claudication, amputation and other cardiovascular events. We have now established a simplified method to enhance the effects of therapeutic transgenes by selecting for and transplanting only transduced cells. Herein, mesenchymal stromal cells were transfected to co-express vascular endothelial growth factor as angiogenic factor and enhanced green fluorescent protein as marker. Transfected cells were then collected using flow cytometry based on green fluorescence and transplanted into ischemic hind limbs in mice. Compared with unsorted or untransfected cells, purified cells significantly improved blood perfusion within 21days, suggesting that transplanting only cells that overexpress vascular endothelial growth factor enhances therapeutic angiogenesis. Importantly, this approach may prove to be useful in cell-based gene therapy against a wide spectrum of diseases, simply by replacing the gene to be delivered or the cell to be transplanted.

COA-Cl (2-Cl-C.OXT-A) can promote coronary collateral development following acute myocardial infarction in mice

2-Cl-C.OXT-A (COA-Cl) is a novel nucleic acid analogue that promotes tube-forming activity of human umbilical vein endothelial cells (HUVEC) through vascular endothelial growth factor (VEGF). The development of coronary collateral circulation is critical to rescue the ischemic myocardium and to prevent subsequent irreversible ischemic injury. We evaluated whether COA-Cl can promote angiogenesis in ischemic tissue, reduce infarct size and preserve cardiac contractility in vivo. Mice received COA-Cl or placebo daily for three days after myocardial infarction (MI) by coronary ligation. The degree of angiogenesis in ischemic myocardium was assessed by staining endothelial cells and vascular smooth muscle cells, and measuring infarct size/area-at-risk. In mice treated with COA-Cl, enhanced angiogenesis and smaller infarct size were recognized, even given a similar area at risk. We observed increases in the protein expression levels of VEGF and in the protein phosphorylation level of eNOS. In addition, the heart weight to body weight ratio and myocardial fibrosis in COA-Cl mice were decreased on Day 7. Administration of COA-Cl after MI promotes angiogenesis, which is associated with reduced infarct size and attenuated cardiac remodeling. This may help to prevent heart failure due to cardiac dysfunction after MI.