Femoral Artery Ligation Research Articles

Simultaneous Upregulation of Elastolytic and Elastogenic Factors Are Necessary for Regulated Collateral Diameter Expansion.

Background: During arteriogenesis, outward remodeling of the arterial wall expands luminal diameter to produce increased conductance in developing collaterals. We have previously shown that diameter expansion without loss of internal elastic lamina (IEL) integrity requires both degradation of elastic fibers and LOX-mediated repair. The aim of this study was to investigate the expression of genes involved in remodeling of the extracellular matrix (ECM) using a model of arteriogenesis.Methods: Sprague-Dawley rats underwent femoral artery ligation with distal arteriovenous fistula (FAL + AVF) placement. Profunda femoral arteries (PFA) were harvested for analysis at various time points. Serum desmosine, an amino acid found exclusively in elastin, was evaluated with enzyme-linked immunosorbent assay (ELISA) as a marker of tissue elastolysis. Tissue mRNA isolated from FAL + AVF exposed PFAs was compared to the contralateral sham-operated using qPCR. HCAECs were cultured under low shear stress (8 dyn·s/cm2) for 24 h and then exposed to high shear stress (40 dyn·s/cm2) for 2–6 h. Primers used included FBN-1, FBN-2, Timp-2, LOX-1, Trop-E, Cath-K, Cath-S, MMP-2, MMP-9, FBLN-4, and FBLN-5 and were normalized to GAPDH. mRNA fold changes were quantified using the 2-ΔΔCq method. Comparisons between time points were made with non-parametric ANOVA analysis with Bonferroni adjustment.Results: PFAs showed IEL reorganization during arteriogenesis. Serum desmosine levels are significantly elevated at 2 days and one week, with a return to baseline thereafter (p < 0.01). Expression of ECM structural proteins (FBN-1, FBN-2, FBLN-4, FBLN-5, Tropoelastin, TIMP-2, LOX-1) and elastolytic proteins (MMP-2, MMP-9, Cathepsin S, Cathepsin K) exhibited an early peak (p < 0.05) relative to sham PFAs. After two weeks, expression returned to baseline. HCAECs demonstrated upregulation of FBN-2, FBLN-5, LOX-1 and Trop-E at 4 h of high shear stress, as well as elastolytic protein MMP-2.Conclusions: Elastin degradation begins early in arteriogenesis and is mediated by local upregulation of elastolytic genes. Elastolysis appears to be simultaneously balanced by production of elastic fiber components which may facilitate stabilization of the IEL. Endothelial cells are central to initiation of arteriogenesis and begin ECM remodeling in response to altered shear stress.

Deficiency of lncRNA SNHG12 impairs ischemic limb neovascularization by altering an endothelial cell cycle pathway.

SNHG12, a long noncoding RNA (lncRNA) dysregulated in atherosclerosis, is known to be a key regulator of vascular senescence in endothelial cells (ECs). However, its role in angiogenesis and peripheral artery disease has not been elucidated. Hind-limb ischemia studies using femoral artery ligation (FAL) in mice showed that SNHG12 expression falls readily in the acute phase of the response to limb ischemia in gastrocnemius muscle and recovers to normal when blood flow recovery is restored to ischemic muscle, indicating that it likely plays a role in the angiogenic response to ischemia. Gain- and loss-of-function studies demonstrated that SNHG12 regulated angiogenesis — SNHG12 deficiency reduced cell proliferation, migration, and endothelial sprouting, whereas overexpression promoted these angiogenic functions. We identified SNHG12 binding partners by proteomics that may contribute to its role in angiogenesis, including IGF-2 mRNA–binding protein 3 (IGF2BP3, also known as IMP3). RNA-Seq profiling of SNHG12-deficient ECs showed effects on angiogenesis pathways and identified a strong effect on cell cycle regulation, which may be modulated by IMP3. Knockdown of SNHG12 in mice undergoing FAL using injected gapmeRs) decreased angiogenesis, an effect that was more pronounced in a model of insulin-resistant db/db mice. RNA-Seq profiling of the EC and non-EC compartments in these mice revealed a likely role of SNHG12 knockdown on Wnt, Notch, and angiopoietin signaling pathways. Together, these findings indicate that SNHG12 plays an important role in the angiogenic EC response to ischemia.

Endothelial S1pr2 regulates post-ischemic angiogenesis via AKT/eNOS signaling pathway.

Aims: It is important to understand the mechanism that regulates post-ischemic angiogenesis and to explore a new therapeutic target for an effective improvement of revascularization in peripheral artery disease (PAD) patients. Post-ischemic angiogenesis is a highly orchestrated process, which involves vascular endothelial cells (ECs) proliferation, migration and assembly into capillaries. We found a significant reduction of S1pr2 (sphingosine 1-phosphate receptor 2) in endothelial cells after hindlimb ischemia (HLI). We thus hypothesized that EC-S1pr2 might be involved in the regulation of post-ischemic angiogenesis and blood flow recovery during peripheral arterial disease (PAD).Methods and Results: We generated both EC-specific S1pr2 loss-of-function and S1pr2 gain-of-function mice. Our study showed that EC-specific S1pr2 loss-of-function significantly enhanced post-ischemic angiogenesis and improved blood flow recovery upon femoral artery ligation, whereas the EC-specific S1pr2 gain-of-function severely hindered post-ischemic angiogenesis and reduced blood flow recovery in ischemic limbs. We next identified that S1pr2 inhibited AKT/eNOS signaling pathway, and thus inhibited EC proliferation/migration and angiogenic activity. As expected, pharmacological inhibition of S1pr2 by JTE013 improved post-ischemic angiogenesis and improved blood flow perfusion after femoral artery ligation. Moreover, we developed RGD-peptide magnetic nanoparticles packaging S1pr2-siRNA which specifically targeted ECs and achieved an efficient silencing of S1pr2 expression in ECs in vivo. This EC-targeted strategy to dampen S1pr2 significantly enhanced post-ischemic angiogenesis and boosted blood perfusion after HLI, supplying a novel therapy target for patients with peripheral arterial disease.Conclusions: This present study demonstrates that EC-expressing S1pr2 tightly controls post-ischemic angiogenesis and blood flow perfusion recovery. This research provides a novel strategy for EC-target knockdown of S1pr2 as a new therapeutic intervention for patients with peripheral artery disease.

Loss of Id3 (Inhibitor of Differentiation 3) Increases the Number of IgM-Producing B-1b Cells in Ischemic Skeletal Muscle Impairing Blood Flow Recovery During Hindlimb Ischemia.

Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease. The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Id3 (inhibitor of differentiation 3) in regulating blood flow in a murine model of hindlimb ischemia (HLI). Approach and Results: HLI was modeled through femoral artery ligation and resection and blood flow recovery was quantified by laser Doppler perfusion imaging. Mice with global Id3 deletion had significantly impaired perfusion recovery at 14 and 21 days of HLI. Endothelial- or myeloid cell-specific deletion of Id3 revealed no effect on perfusion recovery while B-cell-specific knockout of Id3 (Id3BKO) revealed a significant attenuation of perfusion recovery. Flow cytometry revealed no differences in ischemia-induced T cells or myeloid cell numbers at 7 days of HLI, yet there was a significant increase in B-1b cells in Id3BKO. Consistent with these findings, ELISA (enzyme-linked immunoassay) demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when endothelial cells were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3BKO mice showed reduced density of total CD31+ and αSMA+CD31+ vessels. This study is the first to demonstrate a role for B-cell-specific Id3 in maintaining blood flow recovery during HLI. Results suggest a role for Id3 in promoting blood flow during HLI and limiting IgM-expressing B-1b cell expansion. These findings present new mechanisms to investigate in peripheral artery disease pathogenesis.

Abstract 13133: Heterogeneous Uptake of [ 99m Tc]microspheres and 201 Tl in Skeletal Muscles in Porcine Model of Hindlimb Ischemia

Introduction: There is a need for quantitative methods to evaluate skeletal muscle blood flow in PAD patients. We assessed skeletal muscle blood flow heterogeneity at rest or vasodilator stress using 99m Tc-microspheres (HSA), that do not cross the capillaries, and 201 Tl, a freely diffusible radiotracer, in a pig hindlimb ischemia model. Methods: Following acute right femoral artery ligation, pigs (n=8) were injected with either 201 Tl or HSA at rest (n=2 per tracer) or under adenosine-stress (n=2 per tracer). Ex vivo SPECT/CT imaging was performed on excised ischemic (I) and non-ischemic (NI) gastrocnemius (GM) and soleus (SM) muscles. SPECT images were analyzed for the ischemic/nonischemic (I/NI) activity ratio, tracer heterogeneity as indicated by the coefficient of variance (CV) of signal intensity and image intensity histograms. Results: The rest 201 Tl I/N ratio for SM was higher (0.86±0.08) compared to stress (0.64±0.16), with no difference in GM. In contrast, the I/N ratio for HSA was near normal in both muscle groups at rest (GM: 0.80±0.21, SM: 1.06±0.45) although decreased during stress (GM: 0.70±0.04, SM: 0.71±0.12). Rest 201 Tl displayed uniformly low CV in both I (GM: 0.43, SM: 0.43) and NI muscles (GM: 0.37, SM: 0.43). Corresponding CV for stress 201 Tl were similar (I - GM: 0.41, SM: 0.42; NI - GM: 0.41, SM: 0.46). Rest HSA CV were higher in I (GM: 0.92, SM: 1.03) and NI (GM: 0.67, SM: 0.69) muscle, although there was greater heterogeneity in the I muscles assessed with HSA. Stress HSA CV decreased in both I (GM: 0.60, SM: 0.73) and NI (GM: 0.52, SM: 0.51). Figure displays representative count histograms. Conclusions: 201 Tl displayed less heterogeneity than HSA. 201 Tl appears to be more sensitive at detecting reduced blood flow at rest, due to changes in microcirculation. There was no change in the I/NI ratio during 201 Tl stress due to capillary recruitment, although HSA did demonstrate a decrease in the I/NI ratio with vasodilation, reflecting proximal vascular occlusion.

Abstract 10451: A Potential Therapeutic Antibody to Induce Collateral Formation in Cardiovascular Diseases

Introduction: Lack of efficient collateralisation during vascular occlusion leads to tissue ischemia in coronary, peripheral and cerebrovascular disease. Patients with peripheral, coronary, and cerebrovascular disease have elevated levels of the anti-angiogenic isoform of VEGF (VEGF-A 165 b), which correlate with poorer outcomes. In experimental animals, VEGF-A 165 b neutralisation improves blood flow and collateralisation after femoral or coronary artery ligation. Hypothesis: We tested the hypothesis that a potent, high affinity, humanised neutralising antibody to VEGF-A 165 b could have therapeutic potential in models of cardiovascular disease. Methods: A mouse monoclonal antibody to VEGF-A 165 b was humanised by cloning and site directed mutagenesis. Multiple clones were screened, and one with high affinity (measured by biolayer interferometry) was amplified. The efficacy in vitro was tested using endothelial cell migration assays and in vivo using the hindlimb ischemia (HLI) model in C57Bl6 mice fed a high fat high sucrose (HFHS) diet, and in Zucker Diabetic Fatty rats. Results: One clone (HC4LC2) had a VEGF-A 165 b affinity of 600pM. 0.3μg/ml of this clone reversed the inhibitory effect of recombinant human VEGF-A 165 b (31±4%, N=6 of VEGF-A 165 a alone) on migration of endothelial cells to 40ng/ml rhVEGF-A 165 a (89±9% N=3). Human monocyte dependent inhibition of VEGF-A 165 a mediated migration was dose dependently reversed by HC4LC2 (EC50, 55ng/ml, N=9 per concentration). HLI in HFHS mice impaired blood flow after 28 days in IgG treated mice (70±7.7% of pre-ischemic blood flow, N=6), which was restored to normal (93.3±5.5, p&lt;0.05, N=8) by intraperitoneal injection of 1mg/kg HC4LC2. Blood flow recovery after HLI was also seen in ZDF rats treated with HC4LC2 (106±16% of pre-ischemic flow, N=5) compared with IgG treated animals (76±.9%, N=5, p&lt;0.05, two-way ANOVA, all values mean ±SEM). Conclusions: These results indicate that HC4LC2 is a humanised, high affinity neutralising antibody to VEGF-A 165 b that could have therapeutic potential for patients with cardiovascular disease, by enhancing collateral formation.

Abstract 12967: Deletion of G Protein-Coupled Receptor 39 Protects Angiogenesis in Hyperglycemia

Introduction: Endothelial cells (ECs) are essential to vascular homeostasis and angiogenesis. Endothelial dysfunction significantly contributes to critical limb ischemia in patients with diabetes. G protein-coupled receptor (GPR) 39 is an orphan receptor whose role in EC functions is unknown. We hypothesize that inhibition of GPR 39 protects angiogenesis under hyperglycemic conditions. Methods and Results: Healthy human aortic endothelial cells (H-HAECs) and diabetic human aortic endothelial cells (D-HAECs) were cultured in vitro . The migration (Modified Boyden Chamber assay) and tube formation capacity of H-HAECs were impaired by the transfection of adenovirus carrying human GPR39 or by a GPR39 agonist, TC-G-1008 (both n=5, p&lt;0.05). Conversely, GPR39 siRNA improved D-HAEC migration (n=4 per group, p&lt;0.05). In an angiogenesis protein profiler array, GPR39 overexpression has significantly reduced vascular endothelial growth factor (VEGF), Interleukin-8, monocyte Chemoattractant Protein-1 (MCP1), and Angiopoietin-2 in H-HAECs (n=5 per group, p&lt;0.05). Adult male GPR39 global knockout (GPR39 KO ) mice and age- and sex-matched wild-type (GPR39 WT ) litters were rendered diet-induced obese (DIO) by a 10-week high-fat and an add-on 8-week hyperglycemia by low-dose streptozotocin (STZ) injections chow as they reflect the human condition where obesity is closely linked to type 2 diabetes development. Hind limb ischemia was created by left femoral artery ligation. Laser Doppler imaging showed that GPR39 KO mice had a faster blood perfusion recovery from ischemia than GPR39 WT mice (n=5 per group, p&lt;0.05). In adult male C57BL/6 with DIO-STZ, intramuscular injection of EC-affinitive AAV-mediated shRNA against GPR39 significantly accelerated blood perfusion recovery and prevented tissue necrosis compared to empty AAV treatment (n=5 per group, p&lt;0.05). Conclusion: Our data indicated that genetic inhibition of GPR39 improved EC functions and accelerated angiogenesis from ischemia. We believe that the investigations in endothelial GPR39 may help better identify the novel therapeutic potential of targeting GPR39 in treating vascular complications in hyperglycemia.

Mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force.

The hypoxia-inducible nuclear-encoded mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) has been demonstrated to decrease oxidative phosphorylation and production of reactive oxygen species in neonatal cardiomyocytes, brain tissue and hypoxic domains of cancer cells. Prolonged local hypoxia can negatively affect skeletal muscle size and tissue oxidative capacity. Although skeletal muscle is a mitochondrial rich, oxygen sensitive tissue, the role of NDUFA4L2 in skeletal muscle has not previously been investigated. Here we ectopically expressed NDUFA4L2 in mouse skeletal muscles using adenovirus-mediated expression and in vivo electroporation. Moreover, femoral artery ligation (FAL) was used as a model of peripheral vascular disease to induce hind limb ischemia and muscle damage. Ectopic NDUFA4L2 expression resulted in reduced mitochondrial respiration and reactive oxygen species followed by lowered AMP, ADP, ATP, and NAD+ levels without affecting the overall protein content of the mitochondrial electron transport chain. Furthermore, ectopically expressed NDUFA4L2 caused a ~20% reduction in muscle mass that resulted in weaker muscles. The loss of muscle mass was associated with increased gene expression of atrogenes MurF1 and Mul1, and apoptotic genes caspase 3 and Bax. Finally, we showed that NDUFA4L2 was induced by FAL and that the Ndufa4l2 mRNA expression correlated with the reduced capacity of the muscle to generate force after the ischemic insult. These results show, for the first time, that mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force. Specifically, induced NDUFA4L2 reduces mitochondrial activity leading to lower levels of important intramuscular metabolites, including adenine nucleotides and NAD+ , which are hallmarks of mitochondrial dysfunction and hence shows that dysfunctional mitochondrial activity may drive muscle wasting.

Mesenchymal stem cells prevent ovariectomy-induced osteoporosis formation in mice through intraosseous vascular remodeling

Mesenchymal stem cells (MSCs) can promote osteogenesis and are a promising therapy for postmenopausal osteoporosis. However, the relationship between improved intraosseous microcirculation and increased bone mass induced by MSCs in postmenopausal osteoporosis remains unclear. After the primary MSCs were characterized, they were transplanted into ovariectomized mice. MSCs transplantation enhanced the trabecular number, trabecular bone volume/total volume, and trabecular bone mineral density in ovariectomized mice. To determine the role of MSCs in vascular repair, mice were subjected to femoral artery ligation. Through laser speckle flowmetry, vascular perfusion and femoral trabecular bone and cortical bone analyses, we determined the effects of MSCs in promoting intraosseous angiogenesis and preventing osteoporosis in mice. MSCs effectively prevented postmenopausal osteoporosis development, which is associated with the involvement of MSCs in reestablishment of microcirculation within the skeleton.

Early onset of aging phenotype in vascular repair by Mas receptor deficiency.

Aging is associated with impaired vascular repair following ischemic insult, largely due to reparative dysfunctions of progenitor cells. Activation of Mas receptor (MasR) was shown to reverse aging-associated vasoreparative dysfunction. This study tested the impact of MasR-deficiency on mobilization and vasoreparative functions with aging. Wild type (WT) or MasR-deficient mice (MasR-/- or MasR+/-) at 12-14weeks (young) or middle age (11-12months) (MA) were used in the study. Mobilization of lineage-negative, Sca-1-positive cKit-positive (LSK) cells in response to G-CSF or plerixafor was determined. Hindlimb ischemia (HLI) was induced by femoral artery ligation. Mobilization and blood flow recovery were monitored post-HLI. Radiation chimeras were made by lethal irradiation of WT or MasR-/- mice followed by administration of bone marrow cells from MasR-/- or WT mice, respectively. Nitric oxide (NO) generation by stromal-derived factor-1α (SDF) and mitochondrial reactive oxygen species (mitoROS) levels were determined by flow cytometry. Effect of A779 treatment on mobilization, blood flow recovery, and NO and ROS levels were determined in young WT and MasR+/- mice. Circulating LSK cells in basal or in response to plerixafor or G-CSF or in response to ischemic injury were lower in MasR-/- mice compared to the WT. Responses in MasR+/- mice were similar to the WT at young age but at the middle age, impairments were observed. Impaired mobilization to ischemia or G-CSF was rescued in WT → MasR-/- chimeras. NO levels were lower and mitoROS were higher in MasR-/- LSK cells compared to WT cells. A779 precipitated dysfunctions in young-MasR+/- mice similar to that observed in MA-MasR+/-, and this accompanied decreased NO generation by SDF and enhanced mitoROS levels. This study shows that mice at MA do not exhibit vasoreparative dysfunction. Either partial or total loss of MasR precipitates advanced-aging phenotype likely due to lack of NO and oxidative stress.

MicroRNA-126 mimic administration accelerates vascular perfusion recovery and angiogenesis in a hind limb ischemia model

Abstract Background Peripheral arterial disease caused mainly by atherosclerosis portent significant morbidity, adverse prognosis and mortality, with localized treatment approaches aiming at symptom alleviation and improvement of circulation. Recently, scientific interest has been shifted towards epigenomics, with microRNAs appearing as a future therapeutic target in ischemic cardiovascular diseases due to their potential in regulating angiogenesis. Purpose We investigated the pro-angiogenic effect of miRNA-126 mimic in an in vivo model of hind limb ischemia. Methods Ten-week-old male C57Bl/6 mice (n=20) were subjected to left femoral artery ligation and were treated with microRNA-126 mimic at a dose of 5mg/kg (Group A, n=10) or 0.2ml normal saline (Group B, n=10) on days 1, 3 and 7. Laser Doppler imaging was performed to verify successful ligation on day 0 and to evaluate differences in the ischemic-to-normal (I/N) hind limb perfusion ratio on day 7 and 28. Muscle tissue expression of microRNA-126 and vascular endothelial growth factor (VEGF) was determined via PCR. Results Following microRNA-126 mimic administration in Group A subjects, we noted a qualitative and quantitative stepwise increase in I/N hind limb perfusion ratio [Day 0: 0.354 (0.276, 0.455) vs. Day 8: 0.775 (0.700, 0.844) vs. Day 28: 0.681 (0.660, 0.896), p=0.001] (Figure 1, Panels A and B). In Group B a stepwise increase of lesser magnitude was observed in I/N hind limb perfusion ratio [Day 0: 0.267 (0.164, 0.383) vs. Day 8: 0.400 (0.338, 0.418) vs. Day 28: 0.539 (0.483, 0.603), p=0.074]. Importantly, over time changes of I/N hind limb perfusion ratio were significantly higher in group A compared to group B (p for interaction=0.005) (Figure 1, Panel B). Muscle tissue expression of microRNA-126 in the ischemic hind limb of Group A was 350-fold lower compared to the ischemic hind limb of Group B (p&amp;lt;0.001) (Figure 1, Panel C). A higher expression (14.2-fold) of VEGF in the ischemic hind limb of microRNA-126-treated mice compared to that of control group was detected (p&amp;lt;0.001) (Figure 1, Panel C). A statistically significant negative correlation was noted between microRNA-126 and VEGF tissue expression levels in the ischemic limbs of both Group A and B subjects whereas no correlation between microRNA-126 and VEGF was observed in the non-ischemic hind limbs of the entire study population (Figure 1, Panel D). Conclusion MicroRNA-126 mimic delivery in the ischemic hind limb of mice can accelerate vascular perfusion recovery via angiogenesis, which is mediated by VEGF expression. Funding Acknowledgement Type of funding sources: None. Figure 1

A comparison of acute mouse hindlimb injuries between tourniquet- and femoral artery ligation-induced ischemia-reperfusion

The tourniquet or femoral artery ligation is widely used to stop extremity hemorrhage or create a bloodless operating field in the combat scenario and civilian setting. However, these procedures with subsequent reperfusion also induce ischemia-reperfusion (IR) injuries. To fully evaluate animal models of limb IR injuries, we compared tourniquet- and femoral artery ligation-induced IR injuries in the hindlimb of mice. In C57/BL6 mice, 3 h of unilateral hindlimb ischemia was induced by placement of a rubber band at the hip joint or a surgical ligation of the femoral artery. The tourniquet or femoral artery ligation was then released, allowing for 24 h of reperfusion. Compared to the femoral artery ligation/IR, the tourniquet/IR induced more severe skeletal muscle damage, including muscle necrosis and interruption of muscle fibers. There was no gastrocnemius muscle contraction in tourniquet/IR, while femoral artery ligation/IR markedly weakened gastrocnemius muscle contraction. Motor nerve terminals disappeared, and endplate potentials (EPPs) were undetectable in tourniquet/IR, whereas femoral artery ligation/IR only induced mild impairment of motor nerve terminals and decreased the amplitude of EPPs. Additionally, western blot data showed that proinflammatory cytokine levels (IL-1β and TNF-α) were higher in the tourniquet/IR than that in femoral artery ligation/IR. Moreover, tourniquet/IR caused significant tissue edema and dilation of lymphatic vessels in the hindlimb, compared to femoral artery ligation/IR. The above data demonstrated that tourniquet/IR-induced acute hindlimb injuries are more severe than those induced by femoral artery ligation/IR. This suggests that future investigators should determine which hindlimb IR model (tourniquet/IR or femoral artery ligation/IR) is optimal depending on the purpose of their study.

Abstract MP27: Endothelial Nogo-B Modulates Ischemia Induced Collateral Artery Remodeling

Nogo-B, a member of the reticulon 4 family of proteins, is the dominant isoform expressed in endothelial cells (EC). We have shown that Nogo-B is necessary for blood flow recovery in ischemia. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. However, whether endothelial Nogo-B regulates arteriogenesis and/or angiogenesis is unknown. We generated an inducible EC-specific mouse overexpressing Nogo-B (Ng ECtg ) and investigated the arteriogenesis and angiogenesis in limb ischemia. Ischemia increased endothelial and serum Nogo-B expression. Blood flow recovery was markedly diminished after femoral artery ligation (FAL) in Ng ECtg compared to WT mice, in association with lower collateral density accessed by micro-CT arteriography. There was no reduction in capillary density or decrease in smooth muscle/pericyte and macrophage recruitment in Ng ECtg mice, suggesting EC Nogo-B overexpression regulates arteriogenesis but not angiogenesis. While Ng ECtg mice have normal number of native collateral artery, the early remodeling of the collateral artery was impaired after ischemia. Furthermore, in line with impaired remodeling, Ng ECtg mice have decreased functional (exercise) hyperemia response in the non-ischemic limb in vivo, and resistant arteries from Ng ECtg have diminished flow induced vasodilatation ex vivo compared to WT mice. Moreover, endothelial reconstitution of Nogo-B in global Nogo knockout background (NogoKO ECrc ) restored blood flow recovery after ischemia in vivo, further suggesting the EC specific function of Nogo-B in arteriogenesis. Mechanistically, we have shown that EC isolated from Ng ECtg mice have decreased nitric oxide release. Nogo-B overexpression negatively regulates eNOS phosphorylation, expression and golgi localization in EC. Future parabiosis study is warranted to elucidate the contribution of paracrine or EC intrinsic Nogo-B in modulating arteriogenesis and angiogenesis. In conclusion, our data showed for the first time that endothelial Nogo-B plays important roles in regulating collateral artery remodeling and blood flow through, at least in part, regulating NO bioavailability.

The Absence of Extracellular Cold-Inducible RNA-Binding Protein (eCIRP) Promotes Pro-Angiogenic Microenvironmental Conditions and Angiogenesis in Muscle Tissue Ischemia.

Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31+/CD45−/BrdU+). This was accompanied by a reduction of total leukocyte count (CD45+), neutrophils (MPO+), neutrophil extracellular traps (NETs) (MPO+CitH3+), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68+/MRC1−) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68+/MRC1+) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.

The Effect of MicroRNA-126 Mimic Administration on Vascular Perfusion Recovery in an Animal Model of Hind Limb Ischemia.

Background: MicroRNAs have been linked to angiogenesis and could prove to be valuable future therapeutic targets in ischemic cardiovascular diseases.Methods: Ten-week-old male C57Bl/6 mice were subjected to left femoral artery ligation and were treated with microRNA-126 mimic at a dose of 5 mg/kg (Group A, n = 10) or 5 mg/kg microRNA mimic negative control (Group B, n = 10) on days 1, 3, and 7. Laser Doppler imaging was performed to verify successful ligation on day 0 and to evaluate differences in the ischemic-to-normal (I/N) hind limb perfusion ratio on day 28. Muscle tissue expression of microRNA-126 and vascular endothelial growth factor (VEGF) was determined via PCR.Results: Following microRNA-126 mimic administration in Group A subjects, we noted a stepwise increase in I/N hind limb perfusion ratio (Day 0: 0.364 ± 0.032 vs. Day 8: 0.788 ± 0.049 vs. Day 28: 0.750 ± 0.039, p = 0.001). In Group B a stepwise increase in I/N hind limb perfusion ratio was observed (Day 0: 0.272 ± 0.057 vs. Day 8: 0.382 ± 0.020 vs. Day 28: 0.542 ± 0.028, p = 0.074). Muscle tissue expression of microRNA-126 in the ischemic hind limb of Group A was 350-fold lower compared to the ischemic hind limb of Group B (p < 0.001). A higher expression (14.2-fold) of VEGF in the ischemic hind limb of microRNA-126-treated mice compared to that of control group was detected (p < 0.001). A statistically significant negative correlation was noted between microRNA-126 and VEGF tissue expression levels in the ischemic limbs of the entire study population.Conclusion: MicroRNA-126 delivery in the ischemic hind limb of mice improved vascular perfusion with VEGF upregulation.

Ischaemic tissue released microvesicles induce monocyte reprogramming and increase tissue repair by a tissue factor-dependent mechanism.

Despite increasing evidence that monocytes may acquire endothelial features, it remains unclear how monocytes participate in angiogenesis after ischaemic damage. We investigated whether ischaemic cells can release microvesicles (MVs) and promote neovascularization in a model of peripheral artery disease (PAD). To model PAD, we used an in vivo experimental model of hind-limb ischaemia (HLI) in mice. MVs were isolated from the ischaemic muscle and from peripheral blood at different times after unilateral femoral artery ligation. MVs were phenotypically characterized to identify cell origin. HLI in mice induced the release of MVs with a much higher content of tissue factor (TF) than non-HLI control mice both in the MVs isolated from the affected limb muscle area and from blood. MVs were mainly released from endothelial cells (ECs) and induced Mo differentiation to endothelial cell-like (ECL) cells. Differentiation to ECL cells encompassed highly strict hierarchical transcription factor activation, initiated by ETS1 activation. MVs secreted by microvascular ECs over-expressing TF (upTF-EMVs), were injected in the ischaemic hind-limb in parallel with control EMVs (from random siRNA-treated cells) or EMVs released by silenced TF ECs. In animals treated with upTF-EMVs in the ischaemic zone, there was a highly significant increase in functional new vessels formation (seen by magnetic resonance angiography), a concomitant increase in the pool of circulating Ly6Clow Mo expressing vascular EC markers, and a significantly higher number of Mo/macrophages surrounding and integrating the newly formed collaterals. Ischaemia-activated ECs release EMVs rich in TF that induce monocyte differentiation into ECL cells and the formation of new vessels in the ischaemic zone. TF by this mechanism of formation of new blood microvessels can contribute to ischaemic tissue repair.

RETRACTED ARTICLE: GDF11 enhances therapeutic functions of mesenchymal stem cells for angiogenesis

BackgroundThe efficacy of stem cell therapy for ischemia repair has been limited by low cell retention rate. Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β super family, which has multiple effects on development, physiology and diseases. The objective of the study is to investigate whether GDF11 could affect the efficacy of stem cell transplantation.MethodsWe explored the effects of GDF11 on proangiogenic activities of mesenchymal stem cells (MSCs) for angiogenic therapy in vitro and in vivo.ResultsMouse bone marrow-derived MSCs were transduced with lentiviral vector to overexpress GDF11 (MSCGDF11). After exposed to hypoxia and serum deprivation for 48 h, MSCGDF11 were significantly better in viability than control MSCs (MSCvector). MSCGDF11 also had higher mobility and better angiogenic paracrine effects. The cytokine antibody array showed more angiogenic cytokines in the conditioned medium of MSCGDF11 than that of MSCvector, such as epidermal growth factor, platelet-derived growth factor-BB, placenta growth factor. When MSCs (1 × 106 cells in 50 μl) were injected into ischemic hindlimb of mice after femoral artery ligation, MSCGDF11 had higher retention rate in the muscle than control MSCs. Injection of MSCGDF11 resulted in better blood reperfusion and limb salvage than that of control MSCs after 14 days. Significantly more CD31+ endothelial cells and α-SMA + smooth muscle cells were detected in the ischemic muscles that received MSCGDF11. The effects of GDF11 were through activating TGF-β receptor and PI3K/Akt signaling pathway.ConclusionOur study demonstrated an essential role of GDF11 in promoting therapeutic functions of MSCs for ischemic diseases by enhancing MSC viability, mobility, and angiogenic paracrine functions.

Recovery of limb perfusion and function after hindlimb ischemia is impaired by arterial calcification.

Medial artery calcification results from deposition of calcium hydroxyapatite crystals on elastin layers, and osteogenic changes in vascular smooth muscle cells. It is highly prevalent in patients with chronic kidney disease, diabetes, and peripheral artery disease (PAD), and when identified in lower extremity vessels, it is associated with increased amputation rates. This study aims to evaluate the effects of medial calcification on perfusion and functional recovery after hindlimb ischemia in rats. Medial artery calcification and acute limb ischemia were induced by vitamin D3 (VitD3) injection and femoral artery ligation in rats. VitD3 injection robustly induced calcification in the medial layer of femoral arteries in vivo. Laser Doppler perfusion imaging revealed that perfusion decreased and then partially recovered after hindlimb ischemia in vehicle‐injected rats. In contrast, VitD3‐injected rats showed markedly impaired recovery of perfusion following limb ischemia. Accordingly, rats with medial calcification showed worse ischemia scores and delayed functional recovery compared with controls. Immunohistochemical and histological staining did not show differences in capillary density or muscle morphology between VitD3‐ and vehicle‐injected rats at 28 days after femoral artery ligation. The evaluation of cardiac and hemodynamic parameters showed that arterial stiffness was increased while cardiac function was preserved in VitD3‐injected rats. These findings suggest that medial calcification may contribute to impaired perfusion in PAD by altering vascular compliance, however, the specific mechanisms remain poorly understood. Reducing or slowing the progression of arterial calcification in patients with PAD may improve clinical outcomes.

IL-6 signaling pathway contributes to exercise pressor reflex in rats with femoral artery occlusion in association with Kv4 activity in muscle afferent nerves.

Interleukin‐6 (IL‐6) via trans‐signaling pathway plays a role in modifying muscle sensory nerve‐exaggerated exercise pressor reflex in rats with ligated femoral arteries, but the underlying mechanisms are poorly understood. It is known that voltage‐gated potassium channel subfamily member Kv4 channels contribute to the excitabilities of sensory neurons and neuronal signaling transduction. Thus, in this study, we determined that 1) IL‐6 regulates the exaggerated exercise pressor reflex in rats with peripheral artery disease (PAD) induced by femoral artery ligation and 2) Kv4 channels in muscle dorsal root ganglion (DRG) neurons are engaged in the role played by IL‐6 trans‐signaling pathway. We found that the protein levels of IL‐6 and its receptor IL‐6R expression were increased in the DRGs of PAD rats with 3‐day of femoral artery occlusion. Inhibition of muscle afferents’ IL‐6 trans‐signaling pathway (gp130) by intra‐arterial administration of SC144, a gp130 inhibitor, into the hindlimb muscles of PAD rats alleviated blood pressure response to static muscle contraction. On the other hand, we found that 3‐day femoral occlusion decreased amplitude of Kv4 currents in rat muscle DRG neurons. The homo IL‐6/IL‐6Rα fusion protein (H. IL‐6/6Rα), but not IL‐6 alone significantly inhibited Kv4 currents in muscle DRG neurons; and the effect of H. IL‐6/6Rα was largely reverted by SC144. In conclusion, our data suggest that via trans‐signaling pathway upregulated IL‐6 in muscle afferent nerves by ischemic hindlimb muscles inhibits the activity of Kv4 channels and thus likely leads to adjustments of the exercise pressor reflex in PAD.

Chronic intermittent hypobaric hypoxia attenuates ischemic limb injury by promoting angiogenesis in mice.

This study aimed to evaluate the protective effect of chronic intermittent hypobaric hypoxia (CIHH) against limb ischemic injury. C57BL/6 mice were randomly divided into three groups: limb ischemic injury group (Ischemia, induced by ligation and excision of the left femoral artery), limb ischemia following CIHH pretreatment group (CIHH+Ischemia, simulated a 5000 m altitude hypoxia, 6h per day for 28days, before induction of hind-limb ischemia), and sham group (Sham). The blood flow in the mouse models of hind-limb ischemia was examined using laser doppler imaging. The functional and morphological performance of ischemic muscle was evaluated using contraction force and hematoxylin-eosin and Masson's trichrome staining. Angiogenesis was determined by immunohistochemistry staining of the endothelial markers CD31 and CD34. The protein expressions of angiogenesis-related genes were detected using Western blot assay. Chronic ischemia resulted in reduced blood perfusion, decreased contraction tension, and morphological destruction in gastrocnemius muscle. CIHH pretreatment increased the contractile force and muscle fiber diameter and decreased necrosis and fibrosis of the ischemic muscle. Also, CIHH significantly increased the density of CD31+ and CD34+ cells and promoted the expression of angiogenesis-related molecules in ischemic muscle. These data demonstrate that CIHH has a protective effect against chronic limb ischemia by promoting angiogenesis.