Femoral Artery Ligation Research Articles

Increasing nitric oxide bioavailability fails to improve collateral vessel formation in humanized sickle cell mice

AbstractSickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5–7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5–7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4–5), increased necrosis score (P < 0.05, n = 4–5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4–5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery.

Cobra Venom Factor Boosts Arteriogenesis in Mice.

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Prophylactic exercise-derived circulating exosomal miR-125a-5p promotes endogenous revascularization after hindlimb ischemia by targeting endothelin converting enzyme 1.

BackgroundProphylactic exercise improves clinical outcomes in patients experiencing severe ischemic diseases. Previous studies have shown that exercise could alter the amount or content of circulating exosomes. However, little is known about the role of precursory exercise-derived circulating exosomes (Exe-Exo) in ischemic diseases. We therefore aimed to explore the function and mechanism of Exe-Exo in endogenous revascularization and perfusion recovery in peripheral arterial disease.Methods and ResultsWe first determined that 4 weeks of precursory treadmill exercise improved perfusion recovery on days 7, 14 and 21 after unilateral femoral artery ligation (FAL) but had no effect immediately after ligation. Then, local muscle delivery of Exe-Exo promotes arteriogenesis, angiogenesis and perfusion recovery, which could be abolished by GW4869, a well-recognized pharmacological agent inhibiting exosome release. This suggests that Exe-Exo mediated exercise-induced revascularization. In vitro, Exe-Exo enhanced endothelial cell proliferation, migration and tube formation. In addition, we identified miR-125a-5p as a novel exerkine through exosomal miRNA sequencing and RT-qPCR validation. Inhibition of miR-125a-5p abrogated the beneficial effects of Exe-Exo both in vivo and in vitro. Mechanistically, these exercise-afforded benefits were attributed to the exosomal miR-125a-5p downregulation of ECE1 expression and the subsequent activation of the AKT/eNOS downstream signaling pathway. Specifically, skeletal muscle may be a major tissue source of exercise-induced exosomal miR-125a-5p via fluorescence in situ hybridization.ConclusionsEndogenous circulating exosomal miR-125a-5p promotes exercise-induced revascularization via targeting ECE1 and activating AKT/eNOS downstream signaling pathway. Identify exosomal miR-125a-5p as a novel exerkine, and highlight its potential therapeutic role in the prevention and treatment of peripheral arterial disease.

Femoral artery ligature for treatment of infected groin pseudoaneurysm in injected drug abusers.

Drug addiction is a major social and medical concern. Infected groin pseudoaneurysm (IGP) is the result of direct arterial needlestick injury associated with contamination of the arterial wall or peri-arterial area by the injection equipment. Femoral artery (FA) ligation with extensive debridement is an alternative to direct revascularization in an area of sepsis. In case of femoral bifurcation free of infection or in case of isolated FA below the femoral artery of thigh involvement, a simple ligation of the FA is performed. Ligation of each femoral vessel is indicated in case of extension of the infection to the femoral bifurcation. Proximal ligation is performed on the proximal part of the FA. Distal ligation is performed on the proximal part of the deep artery of thigh and the FA below the origin of the deep artery of thigh. Ligation is effective and represents an appropriate method to control hemorrhage and sepsis syndrome in IGP.

Mesenchymal Stromal Cells Combined With Elastin-Like Recombinamers Increase Angiogenesis In Vivo After Hindlimb Ischemia.

Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)–based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti–human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti–CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.

Soluble guanylate cyclase stimulator praliciguat promotes ischemic leg reperfusion in db/db mice

Lower-limb peripheral artery disease (PAD) is a prevalent and challenging complication of diabetes, requiring innovating therapies. Praliciguat is an orally available stimulator of soluble guanylate cyclase (sGC) reported to have favorable effects on metabolic and hemodynamic endpoints in preclinical and clinical studies, suggesting the potential for benefit in PAD. We evaluated the effect of praliciguat on hindlimb ischemia recovery in a mouse model of diabetes and investigated the molecular mechanism of sGC stimulation. Hindlimb ischemia was induced in db/db mice by ligation and excision of the left femoral artery. Praliciguat 10 mg/kg/day ( n = 10 mice) or vehicle ( n = 10) were administered in the diet for 31 days starting 3 days before surgery. Foot perfusion was assessed with a Laser Doppler Imager and reported as a ratio in the ischemic versus non-ischemic limb. Ischemic leg function was assessed with a 4-point scale: 0, plantar/toe flexion in response to tail traction; 1, plantar but not toe flexion; 2, no flexion; 3, foot dragging. Ischemic foot perfusion and function were better 28 days after surgery in praliciguat than in vehicle treated mice (mean ± SD: perfusion 1.05 ± 0.23 vs. 0.38 ± 0.16; P < 0.0001; function 1.14 ± 0.36 vs. 1.73 ± 0.80; P = 0.03). Praliciguat did not impact angiogenesis. Arteriolar diameter was higher (9.88 ± 0.96 vs. 8.57 ± 0.64 μm; P = 0.004) and ICAM-1 expression lower (186 ± 41 vs. 403 ± 177% of healthy muscle; P = 0.005) in the ischemic limb of mice treated with praliciguat than with vehicle. Praliciguat attenuated accumulation of oxidative pro-angiogenic and pro-inflammatory muscle fibers. In cultured myoblasts praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression. Conditioned medium from praliciguat-treated myoblast decreased ICAM-1 mRNA expression in HUVECs. These results suggest that praliciguat restored perfusion and function in the ischemic muscle of db/db mice by increasing arteriolar diameter and decreasing ICAM-1 expression in endothelial cells via downregulation of Cxcl12 in myocytes.

450-P: Soluble Guanylate Cyclase Stimulator Praliciguat Promotes Ischemic Leg Reperfusion in db/db Mice

Background: Lower-limb peripheral artery disease (PAD) is a prevalent and challenging complication of diabetes, requiring innovating therapies. Praliciguat is an orally available stimulator of soluble guanylate cyclase reported to have favorable effects on metabolic and hemodynamic endpoints in preclinical and clinical studies, suggesting the potential for benefit in PAD. We evaluated the effect of praliciguat on hindlimb ischemia recovery in a mouse model of diabetes. Methods: Hindlimb ischemia was induced in db/db mice by ligation and excision of the left femoral artery. Praliciguat 10mg/kg/day (N=mice) or vehicle (N=10) were administered in the diet for 31 days. Foot perfusion was assessed with a Laser Doppler Imager and reported as a ratio in the ischemic versus non-ischemic limb. Ischemic leg function was assessed with a 4-point scale: 0, plantar/toe flexion in response to tail traction; 1, plantar but not toe flexion; 2, no flexion; 3, foot dragging. Results: Ischemic foot perfusion and function were better 28 days after surgery in praliciguat than in vehicle treated mice (mean ± SD: perfusion 1.± 0.23 vs. 0.38 ± 0.16; p&amp;lt;0.0001; function 1.14 ± 0.36 vs. 1.73 ± 0.80; p=0.03) . Praliciguat did not impact angiogenesis. Arteriolar diameter was higher (9.88 ± 0.96 vs. 8.57 ± 0.64 µm; p=0.004) and ICAM-1 expression lower (186 ± 41 vs. 403 ± 177 % of healthy muscle; p=0.005) in the ischemic limb of mice treated with praliciguat than with vehicle. Praliciguat attenuated accumulation of oxidative pro-angiogenic and pro-inflammatory muscle fibers. In cultured myoblasts praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression. Conditioned medium from praliciguat-treated myoblast decreased ICAM-1 mRNA expression in HUVECs. Conclusion: These results suggest that praliciguat restored perfusion and function in the ischemic muscle of db/db mice by increasing arteriolar diameter and decreasing ICAM-1 expression in endothelial cells via downregulation of Cxcl12 in myocytes. Disclosure N.Foussard: None. E.S.Buys: Employee; Cyclerion Therapeutics, Inc. K.Mohammedi: Board Member; Lilly, Novo Nordisk, Sanofi, Research Support; Cyclerion Therapeutics, Inc., Speaker's Bureau; Abbott, AstraZeneca. M.Renault: None.

484-P: Uncoupling of IL-1beta and VEGF-A Signaling Axis Contributes to Impaired Arteriogenesis in the Context of Aging and Diabetes Mellitus

Peripheral artery disease (PAD) caused by atherosclerosis leads to considerable morbidity and mortality throughout the world, in large part, due to tissue damage from both acute and chronic occlusive ischemia. Preclinical studies have identified mechanisms involving bone marrow derived macrophage (BMDM) -dependent angiogenesis in an inflammation suppressed state and we recently defined a novel IL-1beta-dependent transcriptional regulation of the pro-angiogenic isoform of VEGF-A. We sought to understand the impact of diabetes on inflammatory angiogenesis in the context of aging. We hypothesized that the uncoupling of IL-1beta and VEGF-A expression with consequent impairments in angio/arteriogenesis. Control mice at 26-week-old of age or mice with experimental diabetes have reductions of angio/arteriogenesis, using a PAD model of femoral artery ligation that involves macrophage-directed blood flow recovery. Combined aging with chronic diabetes led to further reductions in blood flow recovery consequent to impaired angiogenesis. We also found elevated VEGF-R2 (relative to VEGF-R1) expression in the inflammatory “M1” state which was associated with elevations of IL-1beta and VEGF-A. Interestingly selective inhibition of VEGF-R2 led to reduced VEGF-A expression despite stable IL-1beta levels, suggesting an uncoupling of the relationship between IL-1beta and VEGF-A. Lastly, BMDMs from aged, diabetic mice demonstrated an uncoupling of IL-1beta and VEGF-A expression and VEGF-R2 was decreased in aged, diabetic BMDMs. Defining inflammatory macrophages as key, early drivers of angio/arteriogenesis via IL-1beta and VEGF-A/VEGF-R2 signaling supports a paradigm shift away from inflammation-suppression for adequate healing, allowing for macrophage reprograming strategies that promote appropriate inflammation-dependent healing responses. Disclosure C.S.Mantsounga: None. S.Sharma: None. C.Lee: None. R.Carley: None. G.Choudhary: None. A.R.Morrison: None. Funding National Institute of Health NIGMS (P20GM103652)

Contrast-Enhanced Ultrasound Reveals Partial Perfusion Recovery After Hindlimb Ischemia as Opposed to Full Recovery by Laser Doppler Perfusion Imaging.

Mouse models are critical in developing new therapeutic approaches to treat peripheral arterial disease (PAD). Despite decades of research and numerous clinical trials, the efficacy of available therapies is limited. This may suggest shortcomings in our current animal models and/or methods of assessment. We evaluated perfusion measurement methods in a mouse model of PAD by comparing laser Doppler perfusion imaging (LDPI, the most common technique), contrast-enhanced ultrasound (CEUS, an emerging technique) and fluorescent microspheres (conventional standard). Mice undergoing a femoral artery ligation were assessed by LDPI and CEUS at baseline and 1, 4, 7, 14, 28, 60, 90 and 150 d post-surgery to evaluate perfusion recovery in the ischemic hindlimb. Fourteen days after surgery, additional mice were measured with fluorescent microspheres, LDPI, and CEUS. LDPI and CEUS resulted in broadly similar trends of perfusion recovery until 7 d post-surgery. However, by day 14, LDPI indicated full recovery of perfusion, whereas CEUS indicated ∼50% recovery, which failed to improve even after 5 mo. In agreement with the CEUS results, fluorescent microspheres at day 14 post-surgery confirmed that perfusion recovery was incomplete. Histopathology and photoacoustic microscopy provided further evidence of sustained vascular abnormalities.

Deletion of Myostatin Resolves Myosteatosis and Improves Angiogenesis in Obese Mice

Andrew C. Speese, B.S.1, Caleb A. Padgett M.S.1, Cody L. Rosewater M.S.1, Zachary L. Corley B.S.1, James D. Mintz M.B.A.1, David J. Fulton Ph.D.1,2, and David W. Stepp Ph.D.1,31Vascular Biology Center, 2Department of Pharmacology and Toxicology, and 3Department of Physiology, Medical College of Georgia, Augusta University, Augusta, GAObjectiveTo test the hypothesis that increased muscle mass achieved through deletion of myostatin improves ischemic angiogenesis in obese mice.Methodsdb/db mice, a well‐described model of obesity, were crossed with mice lacking myostatin (MSTN KO), a myokine that negatively regulates muscle differentiation and growth, to generate lean and obese mice with and without elevated muscle mass (MSTN KO). We utilized confocal and electron microscopy (EM) and nuclear magnetic resonance (NMR) spectroscopy to assess morphology and histology in the skeletal muscle (SKM) of db +/‐_MSTN +/‐ mice. Muscle lipid contents were assessed via mass spectrometry. Femoral artery ligation was used to stimulate in vivo angiogenesis in response to ischemia. Gene expression was assessed by RNA‐Seq with mRNA and protein expression follow‐up in isolated gastrocnemius skeletal muscle via RT‐qPCR and western blotting.ResultsAs previously described, myostatin deletion results in significantly increased muscle mass without altering whole‐body mass, fat percentage, or activity levels in obese db/db mice. Blood glucose is markedly improved but plasma lipids remain elevated. In parallel with these improvements, we observed that obesity inhibits and MSTN deletion restores vascularization following hind limb ischemia. Despite persistent plasma lipedema, ectopic lipid deposition in skeletal muscle was largely ameliorated in obese mice lacking myostatin, a reduction largely explained by a decrease in tissue triglyceride levels. EM and confocal microscopy revealed accumulation of lipid in obese muscle cells that was lacking in obese mice with MSTN KO. The resolved deposition occurred predominantly inside muscle cells with fat cell infiltration in external spaces largely unaffected. RNA‐Seq analysis of skeletal muscle revealed marked upregulation of lipogenesis pathways, notably the expression of stearoyl Co‐A desaturase 1 (SCD1), the rate limiting enzyme for the oleate synthesis and subsequent triglyceride production. SCD1 was elevated in muscle from normal obese mice but not those lacking myostatin at both the protein and RNA level.ConclusionIn summary, increased muscle mass in obese mice provides potent protection to vascular and metabolic health. A potent correlate of this improvement is the resolution of myosteatosis in obese, hypermuscular mice. These data suggest that resolution of myosteatosis, independent of muscle mass, may afford similar protection, possibly by targeted deletion or blockade of SCD1.

Abstract 285: Thrombospondin-5 Is Necessary For Males But Not Females In Hindlimb Ischemia Recovery

Introduction: Ameliorating critical limb ischemia (CLI), in part, relies on arteriogenesis and angiogenesis. We have shown thrombospondin-5 (TSP5) is proangiogenic in vitro. The role of TSP5 in CLI and the differences between males and females are unknown. Hypotheses: blood flow recovery, arteriogenesis and angiogenesis after limb ischemia is diminished (1) in TSP5 null mice compared to controls, and (2) in female compared to male TSP5 null mice. Methods: 14-16 week-old wild type (WT; C57Bl6) or TSP5 null male and female mice underwent left femoral artery ligation and transection (n=6-9/group). Laser Doppler data was collected preoperative, post ligation and at sacrifice (day 14). Blood flow recovery was expressed as a ratio of ischemic/non-ischemic limb. Immunohistochemistry was performed using anti-α-smooth-muscle-actin on the vastus lateralis to quantify arteriogenesis and anti-CD31 on the gastrocnemius to quantify angiogenesis. Arteriole and capillary density were determined by vessel counts/5HPF. Data was analyzed by ANOVA with post hoc testing, with p&lt;0.05 being significant. Results: TSP5 null mice had decreased blood flow recovery in males (WT 0.85 ± 0.04 vs TSP5 0.48 ± 0.06, p&lt;0.001) and females (WT 0.63 ± 0.07 vs TSP5 0.36 ± 0.04, p&lt;0.01). No difference in flow recovery was seen between male and female TSP5 nulls (male 0.48 ± 0.06 vs female 0.36 ± 0.04 p&lt;0.14). Arteriogenesis was impaired in male TSP5 null mice (WT 3.91 ± 0.24 vs TSP5 2.6 ± 0.42, p&lt;0.02), but increased in females (WT 2.5 ± 0.26 vs TSP5 4.35 ± 0.73, p&lt;0.01). Angiogenesis was decreased in TSP5 null males (WT 10.9 ± 0.93 vs TSP5 7.03 ± 0.84, p&lt;0.01), but not females (WT 6.1 ± 0.50 vs TSP5 5.6 ± 0.80 p&lt;0.9). In contrast, flow recovery, angiogenesis and arteriogenesis was decreased in WT females compared to WT males (p&lt; 0.05). Conclusions: Our findings suggest TSP5 is relevant to vascular remodeling following hindlimb ischemia with a divergence in mechanism between males and females. TSP5 is necessary in males for arteriogenesis and angiogenesis. TSP5 in females may be supressive of arteriogenesis and has no effect on angiogenesis. Further investigation into these sexually dimorphic adaptive processes may lead to specific targeted CLI treatments.

Abstract 138: Sex Differences In Limb Ischemia Recovery Following Conditional Endothelial Overexpression Of Cept1

Objectives: Choline enthanolamine phosphotransferase 1 ( CEPT1 ), is essential for endothelial de novo lipogenesis (DNL) and is elevated in diseased peripheral arterial segments. Women are more sensitive to diet-induced DNL, and those with diabetes have higher incidence of severe peripheral arterial disease (PAD). We therefore hypothesized that women may have higher CEPT1 content in diseased peripheral arteries and that murine conditional endothelial overexpression of CEPT1 may lead to sex-specific differences in ischemic recovery. Methods: CEPT1 expression was evaluated in diseased peripheral plaque of 7 women and 9 men. A murine endothelial CEPT1 overexpression model was engineered with Cre-induced expression of a CEPT1 transgene inserted in a C57BL/6J background. Endothelial CEPT1 overexpression was evaluated using RT-PCR. Male (n=5) and female (n=5) received Streptozotocin and unilateral femoral artery ligation. Limb perfusion, appearance, and use were then evaluated. Gastrocnemius was stained with H&amp;E, and endothelial Isolectin. Muscle fiber size and microvascular density were analyzed using Student’s t-test. Results: Compared to males, female patients have higher CEPT1 in peripheral segments (n=7, p&lt;0.005; Fig A). Murine endothelial CEPT1 overexpression had a 7-fold increase in expression compared to control (Fig B). Compared to female mice, males demonstrated improved perfusion (p&lt;0.05; Fig C,D), limb function (p&lt;0.05; Fig E), limb appearance (p&lt;0.05; Fig E), muscle fiber size (p&lt;0.05; Fig F,G), but both has adequate muscle microvascular density (Fig F,H). Conclusion: This study reveals a gender difference in peripheral arterial CEPT1 expression and ischemic recovery following overexpression. Differences do not appear to be directly associated with angiogenesis and may be due to underlying DNL metabolic consequences. These findings may have important implications for the prevalence of severe PAD in diabetic women.

Endothelial Mineralocorticoid Receptors Constrain Arteriogenesis and Perfusion Recovery Following Chronic Arterial Ligation in a Sex‐specific Manner

Vascular occlusive diseases are major contributors to cardiovascular morbidity and mortality. Prior work has implicated mineralocorticoid receptor (MR) signaling in perfusion recovery following arterial occlusion, however, cell‐specific MR signaling in the associated angiogenic and arteriogenic responses has not been elucidated. Further, accumulating evidence has revealed sex‐specific involvement of MR signaling in a number of cardiovascular disease states. Accordingly, we examined the hypothesis that MR signaling, specifically endothelial cell (EC) MR signaling, attenuates vascular compensation and perfusion recovery following femoral artery excision and that this effect would be more pronounced in females. Unilateral femoral artery ligation and excision was performed in male and female C57BL/6J mice treated with vehicle or the MR antagonist spironolactone (Spiro; 20 mg/kg/d, sc) as well as in male and female MR Intact and EC MR knockout (KO) mice. Serial assessment of hindlimb perfusion for 28 days following surgery revealed enhanced perfusion recovery following Spiro treatment in female, but not male, mice. Interestingly, Spiro increased gastrocnemius capillary density in both the ischemic and sham limb in males and females. Following perfusion fixation, collateral remodeling was assessed by micro‐computed tomography revealing enhanced collateral expansion following Spiro treatment in female, but not male, mice. Additional mechanistic experiments similarly revealed increased perfusion recovery and enhanced collateral expansion in female, not male, EC MR KO mice independent of capillary density. At 28 days post‐surgery, distribution analysis revealed larger overall arteriolar diameters only in the collateral zone of EC MR KO females. Further analysis revealed enhanced early (7 days post‐surgery) collateral expansion in male and female EC MR KO mice. In female, but not male, EC MR KO mice this was associated with increased CD68+ macrophages and CD206+ ‘M2’‐like macrophages in the collateral zone. Together, these data demonstrate that EC MR signaling constrains early arteriogenic collateralization following arterial excision in both sexes and constrains long‐term collateralization only in females involving limitation of pro‐arteriogenic CD206+ ‘M2’‐like macrophages in the collateral zone. To our knowledge, this is the first report of a role for EC MR signaling in arteriogenesis and highlights potential use of MR blockers in the treatment of vascular occlusive events.

Abstract 335: Macrophage Interleukin-1 Beta Dependent Angiogenesis During Wound Healing

Angiogenesis during wound healing is thought to require an inflammation suppressed state. However, there is growing evidence that early infiltrating inflammatory macrophages may play an important role in setting the stage for angiogenesis. Here we sought to understand the relationship between the potent inflammatory cytokine, IL-1β, and vascular endothelial growth factor-A (VEFG-A) in the context of two experimental models of angiogenesis-dependent healing: skin punch biopsy and hind limb ischemia. We established a macrophage-specific IL-1 β -deletion model ( mIL-1 β ) to study the relationship between IL-1β and VEGF-A in the context of these models. Using a punch biopsy model, wounds were quantified, showing remarked expansion of the wounds in mIL-1 β -deleted mice on day 2 of the healing process, resulting in slower wound closure. Analysis from mIL-1 β -deleted mice confirmed decreased macrophage expression of IL-1β and VEGF-A early at day two post-wounding by punch biopsy. In our second approach, we used femoral artery ligation to demonstrate hind limb ischemia. We hypothesized that mIL-1 β -deleted mice would exhibit reduced blood flow recovery due to impaired angiogenesis. Analysis from mIL-1 β -deleted mice indicates reduced IL-1β and VEGF-A expression. In establishing a pathway of IL-1β-dependent macrophage VEGF-A upregulation in the acute injury state, we examined transcriptional factors STAT3 and NF-κB. Constitutively active STAT3 and IKK-2 (NF-κB activator) appear to increase VEGF-A expression, while inhibition of STAT3 and NF-κB exhibits a dose-dependent reduction of VEGF-A expression. In summary, two models of angiogenesis-dependent healing from injury indicate that macrophage VEGF-A expression is dependent on IL-1β expression during early inflammation. Strategies to alleviate impaired wound healing where VEGF-A levels may not be sufficient could require upregulation of the downstream effectors of IL-1β to restore macrophage VEGF-A expression levels required for adequate angiogenesis.

Depletion of γδ T Cells Leads to Reduced Angiogenesis and Increased Infiltration of Inflammatory M1-like Macrophages in Ischemic Muscle Tissue

γδ T cells, a small subset of T cells in blood, play a substantial role in influencing immunoregulatory and inflammatory processes. The functional impact of γδ T cells on angiogenesis in ischemic muscle tissue has never been reported and is the topic of the present work. Femoral artery ligation (FAL) was used to induce angiogenesis in the lower leg of γδ T cell depleted mice and wildtype and isotype antibody-treated control groups. Gastrocnemius muscle tissue was harvested 3 and 7 days after FAL and assessed using (immuno-)histological analyses. Hematoxylin and Eosin staining showed an increased area of tissue damage in γδ T cell depleted mice 7 days after FAL. Impaired angiogenesis was demonstrated by lower capillary to muscle fiber ratio and decreased number of proliferating endothelial cells (CD31+/BrdU+). γδ T cell depleted mice showed an increased number of total leukocytes (CD45+), neutrophils (MPO+) and neutrophil extracellular traps (NETs) (MPO+/CitH3+), without changes in the neutrophils to NETs ratio. Moreover, the depletion resulted in a higher macrophage count (DAPI/CD68+) caused by an increase in inflammatory M1-like macrophages (CD68+/MRC1−). Altogether, we show that depletion of γδ T cells leads to increased accumulation of leukocytes and M1-like macrophages, along with impaired angiogenesis.

Circular RNA circGSE1 promotes angiogenesis in ageing mice by targeting the miR-323-5p/NRP1 axis.

Age is an important factor in many cardiovascular diseases, in which endothelial cells (ECs) play an important role. Circular RNAs (circRNAs) have been reported in many cardiovascular diseases, but their role in ageing EC-related angiogenesis is unclear. We aimed to identify a functional circRNA that regulates angiogenesis during ageing and explore its specific mechanism. In this study, we searched for differentially expressed circRNAs in old endothelial cells (OECs) and young endothelial cells (YECs) by circRNA sequencing and found that circGSE1 was significantly downregulated in OECs. Our study showed that circGSE1 could promote the proliferation, migration and tube formation of OECs in vitro. In a mouse model of femoral artery ligation and ischemia, circGSE1 promoted blood flow recovery and angiogenesis in the ischemic limbs of ageing mice. Mechanistically, we found that overexpressing circGSE1 reduced miR-323-5p expression, increased neuropilin-1 (NRP1) expression, and promoted proliferation, migration, and tube formation in OECs, while knocking down circGSE1 increased miR-323-5p expression, reduced NRP1 expression, and inhibited proliferation, migration, and tube formation in YECs. During EC ageing, circGSE1 may act through the miR-323-5p/NRP1 axis and promote endothelial angiogenesis in mice. Finally, the circGSE1/miR-323-5p/NRP1 axis could serve as a potential and promising therapeutic target for angiogenesis during ageing.

Bioartificial pulsatile cuffs fabricated from human induced pluripotent stem cell-derived cardiomyocytes using a pre-vascularization technique

There is great interest in the development of techniques to bioengineer pulsatile myocardial tissue as a next-generation regenerative therapy for severe heart failure. However, creation of thick myocardial grafts for regenerative medicine requires the incorporation of blood vessels. In this study, we describe a new method of constructing a vascular network in vivo that allows the construction of thick human myocardial tissue from multi-layered cell sheets. A gelatin sheet pre-loaded with growth factors was transplanted onto the superficial femoral artery and vein of the rat. These structures were encapsulated together within an ethylene vinyl alcohol membrane and incubated in vivo for 3 weeks (with distal superficial femoral artery ligation after 2 weeks to promote blood flow to the vascular bed). Subsequently, six cardiomyocyte sheets were transplanted onto the vascular bed in two stages (three sheets, two times). Incubation of this construct for a further week generated vascularized human myocardial tissue with an independent circulation supplied by an artery and vein suitable for anastomosis to host vessels. Notably, laminating six cell sheets on the vascular bed in two stages rather than one allowed the creation of thicker myocardial tissue while suppressing tissue remodeling and fibrosis. Finally, the pulsatile myocardial tissue was shown to generate auxiliary pressure when wrapped around the common iliac artery of a rat. Further development of this technique might facilitate the generation of circulatory assist devices for patients with heart failure.

Shuxuetong injection and its peptides enhance angiogenesis after hindlimb ischemia by activating the MYPT1/LIMK1/Cofilin pathway

Ethnopharmacological relevanceShuxuetong (SXT) injection is formulated by leech and earthworm, has been widely used in the treatment of thrombotic cardiovascular and cerebrovascular diseases with remarkable clinical efficacy. Aim of the studyThe purpose of this study is to investigate the protective mechanism of SXT injection on the mice model of hindlimb ischemia, and to evaluate the angiogenic effects of SXT injection and its main active substances. Materials and methodsHindlimb ischemia was induced by left femoral artery ligation. After operation, the mice were injected with saline, 10 mg/kg/d cilostazol, 37.5 mg/kg/d SXT injection, 75 mg/kg/d SXT injection and 150 mg/kg/d SXT injection via tail vein for 4 weeks. Ischemia severity was assessed using laser Doppler perfusion imaging system. Tissue recovery and capillary density were evaluated by histological and immunofluorescent staining. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF-BB) expression were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. Human umbilical vein endothelial cells (HUVECs) proliferation was measured using a BrdU kit and the viability of HUVECs was performed by MTT assay. Migration of HUVECs was performed by the wound healing method and a modified transwell assay. Capillary tube formation by HUVECs was examined by using Matrigel assay. Western blotting was used to detect the expressions of p-Cofilin, p-MYPT1, and p-LIMK1. ResultsSXT injection treatment significantly restored the blood flow and reduced tissue injury in mouse gastrocnemius muscle. SXT injection treatment increased capillary density and promoted angiogenesis in hindlimb ischemia. Moreover, SXT injection enhanced the expression of VEGF-A and PDGF-BB at both mRNA and protein levels in ischemic tissue of mice. SXT injection and its main active peptides dramatically increased the migration and capillary tube formation of HUVECs. SXT injection and its peptides enhanced protein expressions of the phosphorylation of MYPT1, Cofilin, and LIMK1. DSYVGDEAQSKR, YNELRVAPEEHP, and IQFLPEGSPVTM may act as the active components of SXT injection. ConclusionSXT injection promoted angiogenesis and improved function recovery in hindlimb ischemia mice by regulation of VEGF-A/PDGF-BB. Moreover, SXT injection and its active peptides induced cell migration and tube formation in HUVECs through activating the MYPT1/LIMK1/Cofilin pathway. This study provided experimental basis for SXT injection in the treatment of ischemic diseases and revealed the effective substance of SXT injection in regulating angiogenesis, providing better evidence for the clinical application of SXT injection.

Collateral Arteriogenesis Involves a Sympathetic Denervation That Is Associated With Abnormal α-Adrenergic Signaling and a Transient Loss of Vascular Tone.

Stimulating collateral arteriogenesis is an attractive therapeutic target for peripheral artery disease (PAD). However, the potency of arteriogenesis-stimulation in animal models has not been matched with efficacy in clinical trials. This may be because the presence of enlarged collaterals is not sufficient to relieve symptoms of PAD, suggesting that collateral function is also important. Specifically, collaterals are the primary site of vascular resistance following arterial occlusion, and impaired collateral vasodilation could impact downstream tissue perfusion and limb function. Therefore, we evaluated the effects of arteriogenesis on collateral vascular reactivity. Following femoral artery ligation in the mouse hindlimb, collateral functional vasodilation was impaired at day 7 (17 ± 3 vs. 60 ± 8%) but restored by day 28. This impairment was due to a high resting diameter (73 ± 4 μm at rest vs. 84 ± 3 μm dilated), which does not appear to be a beneficial effect of arteriogenesis because increasing tissue metabolic demand through voluntary exercise decreased resting diameter and restored vascular reactivity at day 7. The high diameter in sedentary animals was not due to sustained NO-dependent vasodilation or defective myogenic constriction, as there were no differences between the enlarged and native collaterals in response to eNOS inhibition with L-NAME or L-type calcium channel inhibition with nifedipine, respectively. Surprisingly, in the context of reduced vascular tone, vasoconstriction in response to the α-adrenergic agonist norepinephrine was enhanced in the enlarged collateral (−62 ± 2 vs. −37 ± 2%) while vasodilation in response to the α-adrenergic antagonist prazosin was reduced (6 ± 4% vs. 22 ± 16%), indicating a lack of α-adrenergic receptor activation by endogenous norepinephrine and suggesting a denervation of the neuroeffector junction. Staining for tyrosine hydroxylase demonstrated sympathetic denervation, with neurons occupying less area and located further from the enlarged collateral at day 7. Inversely, MMP2 presence surrounding the enlarged collateral was greater at day 7, suggesting that denervation may be related to extracellular matrix degradation during arteriogenesis. Further investigation on vascular wall maturation and the functionality of enlarged collaterals holds promise for identifying novel therapeutic targets to enhance arteriogenesis in patients with PAD.

Notch1 promotes ordered revascularization through Semaphorin 3g modulation of downstream vascular patterning signalling factors.

Here we genetically and functionally addressed potential pathways of Notch signalling in mediating vascular regeneration in mouse models. We first used transgenic adult mice with either gain‐ or loss‐of‐function Notch signalling in vascular endothelial cells and monitored perfusion in the hindlimb following ischaemia induced by femoral artery ligation. Mice deficient in Notch signalling showed defective perfusion recovery and expansion of collateral arteries. Transcriptomics analysis of arterial endothelial cells in the Notch mutants identified the guidance factor Sema3g as a candidate gene mediating reperfusion downstream of Notch. Studies in the retinal circulation showed the central role of SEMA3G downstream of Notch signalling in the orderly regulation of vascular patterning. These studies in multiple vascular beds show the primacy of Notch signalling and downstream generation of guidance peptides such as SEMA3G in promoting well‐ordered vascular regeneration. Key points Notch signalling is a critical mediator of revascularization. Yet the cellular processes activated during recovery following vascular injury are incompletely understood. Here we used genetic and cellular approaches in two different vascular beds and cultured endothelial cells to address the generalizability of mechanisms.By utilizing a highly reproducible murine model of hindlimb ischaemia in transgenic mice in which Notch signalling was inhibited at the transcriptional level, we demonstrated the centrality of Notch signalling in perfusion recovery and revascularization.RNA‐sequencing of Notch mutants identified class 3 Semaphorins regulated by Notch signalling as downstream targets. Studies in retinal vessels and endothelial cells showed an essential role of guidance peptide Sema3g as a modulator of angiogenesis and orderly vascular patterning.The Notch to Sema3g signalling axis functions as a feedback mechanism to sculpt the growing vasculature in multiple beds.