Angiogenesis In Peripheral Arterial Disease Research Articles

Dapagliflozin Improves Angiogenesis after Hindlimb Ischemia through the PI3K-Akt-eNOS Pathway.

Recently, the vascular protective effect of anti-diabetic agents has been receiving much attention. Sodium glucose cotransporter 2 (SGLT2) inhibitors had demonstrated reductions in cardiovascular (CV) events. However, the therapeutic effect of dapagliflozin on angiogenesis in peripheral arterial disease was unclear. This study aimed to explore the effect and mechanism of dapagliflozin on angiogenesis after hindlimb ischemia. We first evaluated the effect of dapagliflozin on post-ischemic angiogenesis in the hindlimbs of rats. Laser doppler imaging was used to detect the hindlimb blood perfusion. In addition, we used immunohistochemistry to detect the density of new capillaries after ischemia. The relevant signaling pathways of dapagliflozin affecting post-ischemic angiogenesis were screened through phosphoproteomic detection, and then the mechanism of dapagliflozin affecting post-ischemic angiogenesis was verified at the level of human umbilical vein endothelial cells (HUVECs). After subjection to excision of the left femoral artery, all rats were randomly distributed into two groups: the dapagliflozin group (left femoral artery resection, receiving intragastric feeding with dapagliflozin (1 mg/kg/d), for 21 consecutive days) and the model group, that is, the positive control group (left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days). In addition, the control group, that is the negative control group (without left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days) was added. At day 21 post-surgery, the dapagliflozin-treatment group had the greatest blood perfusion, accompanied by elevated capillary density. The results showed that dapagliflozin could promote angiogenesis after hindlimb ischemia. Then, the ischemic hindlimb adductor-muscle tissue samples from three rats of model group and dapagliflozin group were taken for phosphoproteomic testing. The results showed that the PI3K-Akt-eNOS signaling pathway was closely related to the effect of dapagliflozin on post-ischemic angiogenesis. Our study intended to verify this mechanism from the perspective of endothelial cells. In vitro, dapagliflozin enhanced the tube formation, migration, and proliferation of HUVECs under ischemic and hypoxic conditions. Additionally, the dapagliflozin administration upregulated the expression of angiogenic factors phosphorylated Akt (p-Akt) and phosphorylated endothelial nitric oxide synthase (p-eNOS), as well as vascular endothelial growth factor A (VEGFA), both in vivo and in vitro. These benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. dapagliflozin could promote angiogenesis after ischemia. This effect might be achieved by promoting the activation of the PI3K-Akt-eNOS signaling pathway. This study provided a new perspective, new ideas, and a theoretical basis for the treatment of peripheral arterial disease.

Abstract 1141: Unveiling Endothelial Cell Metabolic Pathways Il21 Driven Angiogenesis And Barrier Stability In Ischemic Conditions

Background: Angiogenesis is the growth of new blood vessels from preexisting vascular structures. Clinical trials using vascular endothelial growth factor 165a (VEGF165a) for angiogenesis in peripheral arterial disease (PAD) yielded little success. Glycolysis accounts for 85% of ATP production in endothelial cells (ECs) and is further activated during VEGF165a induced angiogenesis. Our previous research demonstrated interleukin 21 (IL21) promoting angiogenesis in PAD models independently of the VEGF165a pathway. Hypothesis: IL21 utilizes different metabolic pathways than glycolysis in ECs under ischemic conditions. Goal: Explore the metabolic pathway(s) activated in ECs in response to IL21 and its functional consequence on angiogenesis in preclinical models of PAD. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured under hypoxia and serum starvation media, treated with PBS (control), VEGF165a (50 ng/mL), or IL21 (50 ng/mL). Methods included qPCR, Western blot (WB) of the genes of the rate controlling enzymes of the main metabolic pathways , angiogenesis assay tube formation on Matrigel and assessing endothelial barrier function using trans endothelial resistance (TER). Results: In HUVECs under HSS, using qPCR, IL21 vs. PBS upregulates glutamine fructose-6-phosphate amido-transferase isoform 1 (GFAT1); the rate limiting enzyme for hexosamine biosynthesis pathway (HBP) (Relative expression 1.28±0.08 SD vs. 1.00±0.04 SD, n=3/group, p=0.03) while VEGF165a did not (Relative expression 1.04±0.03 SD vs. 1.00±0.04 SD, n=3, p=0.23). WB showed similar finding where IL21 vs. PBS increased GFAT1 expression under the same conditions (Relative expression 1.74±0.39 SD vs. 1.00±0.16 SD, n=3/group, p=0.04) while VEGF165a did not. Knocking down GFAT1 reduced IL21 mediated hypoxia dependent angiogenesis (number of tubes, 1.00±0.37 SD vs. 0.37±0.10 SD, n=4-5/group, p<0.0001) but not VEGF165a. The activation of HBP by IL21, was associated with a more stable barrier function compared to VEGF165a (TER relative to control; 0.87±0.01 vs. 0.79±0.01, n=5/group, p<0.0001). Conclusion: These results unmask an unexpected role of HBP in angiogenesis regulation, emphasizing the significance of EC metabolism under PAD conditions.

Epigenetic Regulation of Angiogenesis in Peripheral Artery Disease.

Peripheral arterial disease (PAD) represents a global health concern with a rising prevalence attributed to factors such as obesity, diabetes, aging, and smoking. Among patients with PAD, chronic limb-threatening ischemia (CLTI) is the most severe manifestation, associated with substantial morbidity and mortality. While revascularization remains the primary therapy for CLTI, not all patients are candidates for such interventions, highlighting the need for alternative approaches. Impaired angiogenesis, the growth of new blood vessels, is a central feature of PAD, and despite decades of research, effective clinical treatments remain elusive. Epigenetics, the study of heritable changes in gene expression, has gained prominence in understanding PAD pathogenesis. Here, we explore the role of epigenetic regulation in angiogenesis within the context of PAD, with a focus on long non-coding RNAs and fibroblast-endothelial cell transdifferentiation. Additionally, we discuss the interplay between metabolic control and epigenetic regulation, providing insights into potential novel therapeutic avenues for improving PAD treatments. This review aims to offer a concise update on the application of epigenetics in angiogenesis and PAD research, inspiring further investigations in this promising field.

Correction to: Pentose Pathway Activation Is Superior to Increased Glycolysis for Therapeutic Angiogenesis in Peripheral Arterial Disease.

Correction to: Pentose Pathway Activation Is Superior to Increased Glycolysis for Therapeutic Angiogenesis in Peripheral Arterial Disease.

Pentose Pathway Activation Is Superior to Increased Glycolysis for Therapeutic Angiogenesis in Peripheral Arterial Disease.

Background In endothelial cells (ECs), glycolysis, regulated by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, isoform-3), is the major metabolic pathway for ATP generation. In preclinical peripheral artery disease models, VEGF165a (vascular endothelial growth factor165a) and microRNA-93 both promote angiogenesis. Methods and Results Mice following hind-limb ischemia (HLI) and ECs with, and without, hypoxia and serum starvation were examined with, and without, microRNA-93 and VEGF165a. Post-HLI perfusion recovery was monitored. EC metabolism was studied using seahorse assay, and the expression and activity of major metabolism genes were assessed. Reactive oxygen species levels and EC permeability were evaluated. C57Bl/6J mice generated a robust angiogenic response to HLI, with ECs from ischemic versus nonischemic muscle demonstrating no increase in glycolysis. Balb/CJ mice generated a poor angiogenic response post-HLI; ischemic versus nonischemic ECs demonstrated significant increase in glycolysis. MicroRNA-93-treated Balb/CJ mice post-HLI showed better perfusion recovery, with ischemic versus nonischemic ECs showing no increase in glycolysis. VEGF165a-treated Balb/CJ mice post-HLI showed no improvement in perfusion recovery with ischemic versus nonischemic ECs showing significant increase in glycolysis. ECs under hypoxia and serum starvation upregulated PFKFB3. In ECs under hypoxia and serum starvation, VEGF165a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR-93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA-93 versus VEGF165a upregulated glucose-6-phosphate dehydrogenase expression and activity, activating the pentose phosphate pathway. MicroRNA-93 versus control increased reduced nicotinamide adenine dinucleotide phosphate and virtually eliminated the increase in reactive oxygen species. In ECs under hypoxia and serum starvation, VEGF165a significantly increased and miR-93 decreased EC permeability. Conclusions In peripheral artery disease, activation of the pentose phosphate pathway to promote angiogenesis may offer potential therapeutic advantages.

Abstract 14054: Monocyte Vascular Endothelial Growth Factor Receptor-1 Signaling in Peripheral Artery Disease

Introduction: Peripheral artery disease (PAD) is caused by atherosclerosis of large arteries of the leg(s). Beyond complications from poor perfusion to the legs, patients with PAD suffer higher than expected rates of heart attack and strokes. Monocytes are subdivided into three subtypes: CD14++CD16- (classical), CD14+CD16+ (intermediate), and CD14-CD16+ (non-classical). The intermediate monocyte subtype is linked to PAD. Vascular Endothelial Growth Factor receptor (VEGFR)-1 is a member of tyrosine kinase family that is expressed in monocytes and has high affinity for the VEGF-A ligand. Recent studies showed that inhibition of VEGFR1/STAT-3 signaling decreases angiogenesis in PAD. We hypothesized that in patients with PAD, intermediate monocytes would have less VEGFR1/STAT3 activation than controls. Methods: Blood samples were collected from PAD patients (n=11) and risk factor closely matched controls (n=17). PBMCs were isolated by gradient centrifugation. By flow cytometry, gating strategy on monocytes was done by excluding granulocytes (CD66b), B and T lymphocytes (CD3,CD19), and natural killer cells (CD56). Next, monocytes were subdivided into three populations (intermediate, classical, and non-classical) by gating according to their CD14/CD16 expression. Within each population, levels of p(Y1333)VEGFR1, VEGFR1, pSTAT3, and STAT3 were determined. Results: Data from flow cytometry analysis showed a significant increase in the intermediate monocyte population in PAD vs control (~1.5-fold change; p=0.01). However, there was no significant difference in the non-classical or classical monocytes between PAD and control. Within the intermediate monocytes, P(Y1333) VEGFR1/VEGFR1 was decreased (~2-fold change) and pSTAT3/STAT3 was significantly decreased (~2-fold change) in PAD vs control. In both the non-classical and classical monocytes, there was no significant difference in either phosphorylated receptor between PAD and control. Conclusions: The data confirm the intermediate monocyte is associated with PAD and suggest that VEGFR1/STAT3 signaling is decreased in intermediate monocytes in PAD vs control. Understanding the mechanism of VEGFR1 activation may prove to be a promising therapeutic target for patients with PAD.

Abstract 15292: Il21R-AS1 Long Non-Coding RNA as Potential Regulator in Therapeutic Angiogenesis in Peripheral Arterial Disease

Background: Peripheral arterial disease (PAD) is the major complication of systemic atherosclerosis. The clinical complications are intermittent claudication (IC) or its more severe form, critical limb ischemia (CLI). Potential therapeutic agents, to treat PAD failed in clinical studies. Vascular endothelial growth factor (VEGF) induced significant edema with no clinical benefit. Still today, no medical therapy exists to improve perfusion in PAD. Our lab has been studying the role of the interleukin-21 receptor (IL21R) in therapeutic angiogenesis. IL21 is undergoing clinical studies but its ability to be a therapeutic may be limited. IL21R transcribes a single human long non-coding RNA, the IL21R- antisense 1 (IL21R-AS1), with no mice homolog. Its role in PAD is not established yet. Hypothesis: IL21R-AS1 can serve as an activator of IL21R signaling to mediate hypoxia-induced angiogenesis without increasing vascular permeability. Methods and Results: Plasma from patients with IC, CLI, and non-PAD controls was obtained from whole blood. IL21R-AS1 was elevated in the plasma of patients with CLI. IL21R-AS1 expression was higher in endothelial cells (ECs) under hypoxia (2% O 2 ) serum starvation (HSS), an in vitro model of PAD. To reduce the expression of IL21R-AS1 cells were transfected with gapmeR, and for the overexpression, IL21-AS1 pCMV-TnT plasmid was used. The knockdown of the IL21R-AS1 decreased tube formation in angiogenesis assay performed on growth factor reduced matrigel, while the overexpression of the lncRNA was pro-angiogenic in hypoxic endothelial cells. We detected IL21R-AS1 in the cytoplasm and showed its translocation to the cell surface in HSS. More importantly, in ECs under HSS, we detected IL21R-AS1 in IL21R immunoprecipitation samples; suggesting a direct interaction between IL21R-AS1 and the IL21R. Both IL21 treatment and IL21R-AS1 overexpression increased transendothelial resistance (TER) in hypoxic ECs, in contrast to the permeability induced by VEGF as measured by ECIS (Endothelial Cell Impedance Sensing). Conclusion: Collectively our data suggest that the lncRNA IL21R-AS1 serves as an activator of the IL21R that can mediate angiogenesis but without inducing vascular leakage.

Targeting Anti-Angiogenic VEGF165b-VEGFR1 Signaling Promotes Nitric Oxide Independent Therapeutic Angiogenesis in Preclinical Peripheral Artery Disease Models.

Nitric oxide (NO) is the critical regulator of VEGFR2-induced angiogenesis. Neither VEGF-A over-expression nor L-Arginine (NO-precursor) supplementation has been effective in helping patients with Peripheral Artery Disease (PAD) in clinical trials. One incompletely studied reason may be due to the presence of the less characterized anti-angiogenic VEGF-A (VEGF165b) isoform. We have recently shown that VEGF165b inhibits ischemic angiogenesis by blocking VEGFR1, not VEGFR2 activation. Here we wanted to determine whether VEGF165b inhibition using a monoclonal isoform-specific antibody against VEGF165b vs. control, improved perfusion recovery in preclinical PAD models that have impaired VEGFR2-NO signaling, including (1) type-2 diabetic model, (2) endothelial Nitric oxide synthase-knock out mice, and (3) Myoglobin transgenic mice that have impaired NO bioavailability. In all PAD models, VEGF165b inhibition vs. control enhanced perfusion recovery, increased microvascular density in the ischemic limb, and activated VEGFR1-STAT3 signaling. In vitro, VEGF165b inhibition vs. control enhanced a VEGFR1-dependent endothelial survival/proliferation and angiogenic capacity. These data demonstrate that VEGF165b inhibition induces VEGFR1-STAT3 activation, which does not require increased NO to induce therapeutic angiogenesis in PAD. These results may have implications for advancing therapies for patients with PAD where the VEGFR2-eNOS-NO pathway is impaired.

Effect of Revascularization on Intramuscular Vascular Endothelial Growth Factor Levels in Peripheral Arterial Disease.

Vascular endothelial growth factor (VEGF) is a potent driver of angiogenesis, which may help to relieve ischemia in peripheral arterial disease (PAD). We aimed to investigate the role of intramuscular VEGF in ischemic and non-ischemic skeletal muscle in PAD patients before and after surgical or endovascular revascularization and different stages of PAD. Biopsies of the gastrocnemius and vastus muscles from twenty PAD patients with stenosis or occlusion of the superficial femoral artery were obtained both during revascularization and 8 weeks postoperatively. The gastrocnemius muscle was considered ischemic, while vastus muscle biopsies served as intraindividual controls. The levels of vascular endothelial growth factor in muscle lysates were then determined by ELISA. Preoperative VEGF levels were significantly higher in ischemic muscles compared to the controls (98.07 ± 61.96 pg/mL vs. 55.50 ± 27.33 pg/mL, p = 0.004). Postoperative values decreased significantly (p = 0.010) to 54.83 ± 49.60 pg/mL in gastrocnemius biopsies. No significant change was observed in vastus muscle biopsies, with mean postoperative VEGF values found at 54.16 ± 40.66 pg/mL. Since all patients still had indications for revascularization, impairment of angiogenesis mechanisms can be assumed. More research about angiogenesis in PAD is needed with the ultimate goal to improve conservative treatment.

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.

MiR‑124‑3p regulates angiogenesis in peripheral arterial disease by targeting STAT3.

Peripheral arterial disease (PAD) is the third leading cause of cardiovascular morbidity worldwide, after coronary artery disease and stroke. As endogenous regulators of gene expression, microRNAs (miRs) are implicated in the development and progression of various diseases, including types of cancer, autoimmune diseases and heart diseases. In the present study, the role of miR-124-3p in PAD was investigated. The reverse transcription-quantitative PCR results indicated that the expression levels of miR-124-3p were significantly increased in the ischemic tissue of the hindlimb ischemia (HLI) model and in hypoxic human umbilical vein endothelial cells compared with the corresponding control groups. Proliferation, wound healing and tube formation assays demonstrated the inhibition of miR-124-3p on angiogenesis in vitro and the HLI model indicated the same function of miR-124-3p in vivo. A dual-luciferase reporter revealed STAT3 as the target of miR-124-3p. The expression levels of miR-124-3p in human blood were negatively correlated with ankle-brachial index, which is an index for the evaluation of the severity of PAD. Collectively, the present study indicated that miR-124-3p was a critical regulator of angiogenesis in PAD, and a potential diagnostic, prognostic and therapeutic target for PAD.

The role of lymphangiogenesis in ischemia-induced angiogenesis for peripheral artery disease

The role of lymphangiogenesis in ischemia-induced angiogenesis for peripheral artery disease

Therapeutic Potential of Human iPS Cell-Derived Endothelial and Mural Cells Incorporated with Gelatin Sponge Scaffold in a Murine Hindlimb Ischemia Model

Introduction - To standardize stem cell-based therapeutic angiogenesis for peripheral artery disease, a transplantation strategy to achieve efficient and sustainable therapeutic effect is required. In this study, we have investigated therapeutic effects of human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and vascular mural cells (MCs) incorporated with gelatin sponge (GS) scaffold in a murine hindlimb ischemia model.

Dual specificity phosphatase 5 regulates perfusion recovery in experimental peripheral artery disease.

Peripheral artery disease (PAD) is caused by atherosclerotic occlusions of vessels outside the heart, particularly those of the lower extremities. Angiogenesis is one critical physiological response to vessel occlusion in PAD, but our understanding of the molecular mechanisms involved in angiogenesis is incomplete. Dual specificity phosphatase 5 (DUSP5) has been shown to play a key role in embryonic vascular development, but its role in post-ischemic angiogenesis is not known. We induced hind limb ischemia in mice and found robust upregulation of Dusp5 expression in ischemic hind limbs. Moreover, in vivo knockdown of Dusp5 resulted in impaired perfusion recovery in ischemic limbs and was associated with increased limb necrosis. In vitro studies showed upregulation of DUSP5 in human endothelial cells exposed to ischemia, and knockdown of DUSP5 in these ischemic endothelial cells resulted in impaired endothelial cell proliferation and angiogenesis, but did not alter apoptosis. Finally, we show that these effects of DUSP5 on post-ischemic angiogenesis are a result of DUSP5-dependent decrease in ERK1/2 phosphorylation and p21 protein expression. Thus, we have identified a role of DUSP5 in post-ischemic angiogenesis and implicated a DUSP5-ERK-p21 pathway that may serve as a therapeutic target for the modulation of post-ischemic angiogenesis in PAD.

Long noncoding RNA Alu-mediated p21 transcriptional regulator promotes proliferation, migration, and pipe-formation of human microvascular endothelial cells by sponging miR-126.

Peripheral artery disease (PAD) is a serious hazard to the elderly in the lower extremity atherosclerotic plaque, accompanied by a large number of angiogenesis. Long noncoding RNA Alu-mediated p21 transcriptional regulator (APTR) exerts important functions in promoting cell growth. Therefore, we planned to research the mechanism of APTR in angiogenesis in PAD. CCK-8 assay, flow cytometry analysis, and migration assay were to detect cell viability, apoptosis, and migration respectively. The interaction between APTR and miR-12 was tested through luciferase activity test. In vitro angiogenesis assay was used to test the number of tubular cells. qRT-PCR and Western blot were to test expression of APTR, miR-126, and angiogenesis relative factors. There was spontaneously pipe-formation in HEMC-1 cells under matrigel condition. Knockdown of APTR inhibited cell viability and migration and reduced the number of tubular cells. Further, APTR sponged miR-126 and downregulating miR-126 to promote angiogenesis. Overexpression of APTR promoted cell activity and migration and increased the number of tubular cells via negatively regulating miR-126. APTR could elevate activating phosphatidylinositol 3 kinase/protein kinase B and mitogen extracellular kinase/extracellular signal-regulated kinase signal pathways via negatively regulating miR-126 to promote cell proliferation, migration, and pipe-formation. We researched the mechanism of angiogenesis that APTR elevated proliferation, migration, and pipe-formation via negatively regulating miR-126.

MicroRNA‑93 regulates angiogenesis in peripheral arterial disease by targeting CDKN1A.

MicroRNAs (miRNAs) are considered to be critical mediators of gene expression with respect to tumor progression, although their role in ischemia-induced angiogenesis is poorly characterized, including in peripheral arterial disease (PAD). Furthermore, the underlying mechanism of action of specific miRNAs in PAD remains unknown. Reverse transcription-quantitative polymerase chain reaction analysis revealed that microRNA-93 (miR-93) was significantly upregulated in patients with PAD and in the EA.hy926 endothelial cells in response to hypoxia. Additionally, miRNA (miR)-93 promoted angiogenesis by enhancing proliferation, migration and tube formation. Cyclin dependent kinase inhibitor 1A (CDKN1A), verified as a potential target gene of miR-93, was inhibited by overexpressed miR-93 at the protein and mRNA expression levels. Furthermore, a hind-limb ischemia model served to evaluate the role of miR-93 in angiogenesis in vivo, and the results demonstrated that miR-93 overexpression enhanced capillary density and perfusion recovery from hind-limb ischemia. Taken together, miR-93 was indicated to be a promising target for pharmacological regulation to promote angiogenesis, and the miR-93/CDKN1A pathway may function as a novel therapeutic approach in PAD.

Antiangiogenic VEGF165b Regulates Macrophage Polarization via S100A8/S100A9 in Peripheral Artery Disease.

Atherosclerotic occlusions decrease blood flow to the lower limbs, causing ischemia and tissue loss in patients with peripheral artery disease (PAD). No effective medical therapies are currently available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing vascular endothelial growth factor (VEGF)-A levels, a potent proangiogenic growth factor to induce angiogenesis, and perfusion recovery were not successful. Alternate splicing in the exon-8 of VEGF-A results in the formation of VEGFxxxa (VEGF165a) and VEGFxxxb (VEGF165b) isoforms with existing literature focusing on VEGF165b's role in inhibiting vascular endothelial growth factor receptor 2-dependent angiogenesis. However, we have recently shown that VEGF165b blocks VEGF-A-induced endothelial vascular endothelial growth factor receptor 1 (VEGFR1) activation in ischemic muscle to impair perfusion recovery. Because macrophage-secreted VEGF165b has been shown to decrease angiogenesis in peripheral artery disease, and macrophages were well known to play important roles in regulating ischemic muscle vascular remodeling, we examined the role of VEGF165b in regulating macrophage function in PAD. Femoral artery ligation and resection were used as an in vivo preclinical PAD model, and hypoxia serum starvation was used as an in vitro model for PAD. Experiments including laser-Doppler perfusion imaging, adoptive cell transfer to ischemic muscle, immunoblot analysis, ELISAs, immunostainings, flow cytometry, quantitative polymerase chain reaction analysis, and RNA sequencing were performed to determine a role of VEGF165b in regulating macrophage phenotype and function in PAD. First, we found increased VEGF165b expression with increased M1-like macrophages in PAD versus non-PAD (controls) muscle biopsies. Next, using in vitro hypoxia serum starvation, in vivo pre clinical PAD models, and adoptive transfer of VEGF165b-expressing bone marrow-derived macrophages or VEGFR1+/- bone marrow-derived macrophages (M1-like phenotype), we demonstrate that VEGF165b inhibits VEGFR1 activation to induce an M1-like phenotype that impairs ischemic muscle neovascularization. Subsequently, we found S100A8/S100A9 as VEGFR1 downstream regulators of macrophage polarization by RNA-Seq analysis of hypoxia serum starvation-VEGFR1+/+ versus hypoxia serum starvation-VEGFR1+/- bone marrow-derived macrophages. In our current study, we demonstrate that increased VEGF165b expression in macrophages induces an antiangiogenic M1-like phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGF165b results in S100A8/S100A9-mediated calcium influx to induce an M1-like phenotype that impairs ischemic muscle revascularization and perfusion recovery.

Interleukin-22 receptor 1 upregulation and activation in hypoxic endothelial cells improves perfusion recovery in experimental peripheral arterial disease

ObjectiveInflammation induced by muscle ischemia is involved in tissue repair and perfusion recovery in peripheral arterial disease (PAD) patients. Interleukin (IL)-22 is an inflammatory cytokine discovered in recent years and shows versatile functions; however, its role in PAD remains unknown. Here, we test whether IL-22 and its receptors are involved in angiogenesis in experimental PAD. Methods and resultsBoth IL-22 and its receptor—IL-22 receptor 1(IL-22R1) were upregulated in muscle and endothelial cells after ischemia. In experimental PAD models, blocking IL-22 using IL-22 monoclonal antibody impaired perfusion recovery and angiogenesis; on the other hand, IL-22 treatment improved perfusion recovery. Ischemic muscle tissue was harvested 3 days after experimental PAD for biochemical test, IL-22 antagonism resulted in decreased Signal Transducer and Activator of Transcription (STAT3) phosphorylation, but did not alter the levels of VEGF-A or cyclic guanine monophosphate (cGMP) levels in ischemic muscle. In cultured endothelial cells, IL-22R1 was upregulated under simulated ischemic conditions, and IL-22 treatment increased STAT3 phosphorylation, endothelial cell survival and tube formation. Knock down of IL-22R1 or treatment with STAT3 inhibitor blunted IL-22-induced endothelial cell survival or tube formation. ConculsionIschemia-induced IL-22 and IL-22R1 upregulation improves angiogenesis in PAD by inducing STAT3 phosphorylation in endothelial cells. IL-22R1 may serve as a new therapeutic target for PAD.

MicroRNA-133a impairs perfusion recovery after hindlimb ischemia in diabetic mice.

Objective: Peripheral arterial disease (PAD) patients with diabetes mellitus suffer from impaired neovascularization after ischemia which results in poorer outcomes. MicroRNA (miR)-133a is excessively expressed in endothelial cells under diabetic conditions. Here, we test whether diabetes-induced miR-133a up-regulation is involved in the impaired capability of neovascularization in experimental PAD models. Methods and results: MiR-133a level was measured by quantitative RT-PCR and showed a higher expression level in the ischemic muscle from diabetic mice when compared with nondiabetic mice. Knockdown of miR-133a using antagomir improved perfusion recovery and angiogenesis in experimental PAD model with diabetes day 21 after HLI. On the other hand, overexpression of miR-133a impaired perfusion recovery. Ischemic muscle was harvested day 7 after experimental PAD for biochemical test, miR-133a antagonism resulted in reduced malondialdehyde, and it increased GTP cyclohydrolase 1 (GCH1), and cyclic guanine monophosphate (cGMP) levels. In cultured endothelial cells, miR-133a antagonism resulted in reduced reactive oxygen species level, and it increased tube formation, nitric oxide (NO), and cGMP level. Moreover, miR-133a antagonism-induced angiogenesis was abolished by GCH1 inhibitor. In contrary, miR-133a overexpression impairs angiogenesis and it reduces GCH1, NO, and cGMP levels in nondiabetic models. Conclusion: Diabetes mellitus-induced miR-133a up-regulation impairs angiogenesis in PAD by reducing NO synthesis in endothelial cells. MiR-133a antagonism improves postischemic angiogenesis.

Growth Factors for Angiogenesis in Peripheral Arterial Disease

Background Peripheral artery disease (PAD) is associated with a high clinical and socioeconomic burden. Treatments to alleviate the symptoms of PAD and decrease the risks of amputation and death are a high societal priority. A number of growth factors have shown a potential to stimulate angiogenesis. Growth factors delivered directly (as recombinant proteins), or indirectly (e.g. by viral vectors or DNA plasmids encoding these factors), have emerged as a promising strategy to treat patients with PAD. Objectives To assess the effects of growth factors that promote angiogenesis for treating people with PAD of the lower extremities. Search methods The Cochrane Vascular Information Specialist searched the Specialised Register (June 2016) and CENTRAL (2016, Issue 5). We searched trial registries for details of ongoing or unpublished studies. We also checked the reference lists of relevant publications and, if necessary, tried to contact the trialists for details of the studies. Selection criteria We included randomised controlled trials comparing growth factors (delivered directly or indirectly) with no intervention, placebo or any other intervention not based on the growth factor's action in patients with PAD of the lower extremities. The primary outcomes were limb amputation, death and adverse events. The secondary outcomes comprised walking ability, haemodynamic measures, ulceration and rest pain. Data collection and analysis Two review authors independently selected trials and assessed the risk of bias. We used outcomes of the studies at low risk of bias for the main analysis and of all studies in the sensitivity analyses. We calculated odds ratios (OR) for dichotomous outcomes and mean differences for continuous outcomes with 95% confidence intervals (CI). We evaluated statistical heterogeneity using the I2 statistic and Cochrane's Q test. We conducted meta-analysis for the overall effect and for each growth factor as a subgroup analysis using OR in a fixed-effect model. We evaluated the robustness of the results in a sensitivity analysis using risk ratio (RR) and/or a random-effects model. We also assessed the quality of the evidence for each outcome. Main results We included 20 trials in the review and used 14 studies (on approximately 1400 participants) with published results in the analyses. Six published studies compared fibroblast growth factors (FGF), four studies hepatocyte growth factors (HGF) and another four studies vascular endothelial growth factors (VEGF), versus placebo or no therapy. Six of these studies exclusively or mainly investigated participants with intermittent claudication and eight studies exclusively participants with critical limb ischaemia. Follow-up generally ranged from three months to one year. Two small studies provided some data at 2 years and one of them also at 10 years. The direction and size of effects for growth factors on major limb amputations (OR 0.99, 95% CI 0.71 to 1.38; 10 studies, N = 1075) and death (OR 0.99, 95% CI 0.69 to 1.41; 12 studies, N = 1371) at up to two years are uncertain. The quality of the evidence is low due to risk of bias and imprecision (at one year, moderate-quality evidence due to imprecision). However, growth factors may decrease the rate of any limb amputations (OR 0.56, 95% CI 0.31 to 0.99; 6 studies, N = 415). The quality of the evidence is low due to risk of bias and selective reporting. The direction and size of effects for growth factors on serious adverse events (OR 1.09, 95% CI 0.79 to 1.50; 13 studies, N = 1411) and on any adverse events (OR 1.10, 95% CI 0.73 to 1.64; 4 studies, N = 709) at up to two years are also uncertain. The quality of the evidence is low due to risk of bias and imprecision (for serious adverse events at one year, moderate-quality evidence due to imprecision). Growth factors may improve haemodynamic measures (low-quality evidence), ulceration (very low-quality evidence) and rest pain (very low-quality evidence) up to one year, but they have little or no effect on walking ability (low-quality evidence). We did not identify any relevant differences in effects between growth factors (FGF, HGF and VEGF). Authors' conclusions The results of this review do not support the use of therapy with the growth factors FGF, HGF or VEGF in people with PAD of the lower extremities to prevent death or major limb amputation or to improve walking ability. However, the use of these growth factors may improve haemodynamic measures and decrease the rate of any limb amputations (probably due to preventing minor amputations) with an uncertain effect on adverse events; an improvement of ulceration and rest pain is very uncertain. New trials at low risk of bias are needed to generate evidence with more certainty.