Femoral Artery Ligation Research Articles

Deletion of AT2 Receptor Prevents SHP-1–Induced VEGF Inhibition and Improves Blood Flow Reperfusion in Diabetic Ischemic Hindlimb

Ischemia caused by narrowing of femoral artery is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes mellitus. We have previously reported that the inhibition of the angiogenic response to VEGF (vascular endothelial growth factor) in diabetic mice was associated with the increased expression of SHP-1 (SH2 domain-containing phosphatase 1), a protein that can be activated by the AT2 (angiotensin II type 2) receptor. Deletion of AT2 receptor has been shown to promote angiogenesis within the ischemic muscle. However, the relative impact of AT2 receptor in diabetic condition remains unknown. Nondiabetic and diabetic AT2 null (Atgr2-/Y) mice underwent femoral artery ligation after 2 months of diabetes mellitus. Blood perfusion was measured every week ≤4 weeks post-surgery. Expression of the VEGF, SHP-1, and renin-angiotensin pathways was evaluated. Blood flow in the ischemic muscle of diabetic Atgr2-/Y mice recovered faster and ≤80% after 4 weeks compared with 51% recovery in diabetic control littermates. Diabetic Atgr2-/Y had reduced apoptotic endothelial cells and elevated small vessel formation compared with diabetic Atgr2+/Y mice, as well as increased SHP-1 expression and reduced VEGF receptor activity. In endothelial cells, high glucose levels and AT2 agonist treatment did not change SHP-1, VEGF, and VEGF receptor expression. However, the activity of SHP-1 and its association with the VEGF receptors were increased, causing inhibition of the VEGF action in endothelial cell proliferation and migration. Our results suggest that the deletion of AT2 receptor reduced SHP-1 activity and restored VEGF actions, leading to an increased blood flow reperfusion after ischemia in diabetes mellitus.

Exaggerated cardiovascular responses to treadmill running in rats with peripheral arterial insufficiency.

Patients with atherosclerotic peripheral artery disease have an augmented pressor response to treadmill walking, but the underlying mechanisms remain poorly understood and difficult to isolate because of the confounding presence of numerous cardiovascular risk factors. In the present study, we tested the hypothesis that a chronic deficit in muscle blood flow capacity would be sufficient to trigger an exaggerated pressor response to dynamic exercise. Sprague-Dawley rats (5 male and 5 female) were instrumented with radiotelemetry devices to measure the cardiovascular responses to treadmill running before and after bilateral femoral artery ligation, which has been previously shown to reduce the blood flow capacity of distal hindlimb muscles by >60%. Treadmill running evoked reproducible increases in mean arterial pressure (MAP) and heart rate (HR), which were significantly augmented 3 days after femoral artery ligation in both male rats [ΔMAP: +10 ± 1 (SE) vs. +18 ± 3 mmHg and ΔHR: +94 ± 12 vs. +148 ± 15 beats/min, P < 0.05] and female rats (ΔMAP: +16 ± 3 vs. +30 ± 5 mmHg and ΔHR: +128 ± 20 vs. +178 ± 19 beats/min, P < 0.05). Similar exaggerated MAP and HR responses were observed at repeated intervals between 3 and 65 days postligation. These findings indicate that a chronic deficit in muscle blood flow capacity is an important, persistent cause of the abnormal pressor and cardioaccelerator responses to dynamic exercise in both male and female rats with peripheral arterial insufficiency. NEW & NOTEWORTHY Using radiotelemetry to assess cardiovascular effects of exercise, we showed that femoral artery obstruction in male and female rats is an important, persistent cause of exaggerated pressor and cardioaccelerator responses to treadmill running. This translational model reproduces the abnormal cardiovascular response to exercise seen in patients with peripheral artery disease. Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/treadmill-bp-in-simulated-peripheral-artery-disease/ .

Mitochondrial-Targeted Antioxidant Maintains Blood Flow, Mitochondrial Function, and Redox Balance in Old Mice Following Prolonged Limb Ischemia.

Aging is a major factor in the decline of limb blood flow with ischemia. However, the underlying mechanism remains unclear. We investigated the role of mitochondrial reactive oxygen species (ROS) with regard to limb perfusion recovery in aging during ischemia. We performed femoral artery ligation in young and old mice with or without treatment with a scavenger of mitochondrial superoxide, MitoTEMPO (180 μg/kg/day, from pre-operative day 7 to post-operative day (POD) 21) infusion using an implanted mini-pump. The recoveries of cutaneous blood flow in the ischemic hind limb were lower in old mice than in young mice but were improved in MitoTEMPO-treated old mice. Mitochondrial DNA damage appeared in ischemic aged muscles but was eliminated by MitoTEMPO treatment. For POD 2, MitoTEMPO treatment suppressed the expression of p53 and the ratio of Bax/Bcl2 and upregulated the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in ischemic aged skeletal muscles. For POD 21, MitoTEMPO treatment preserved the expression of PGC-1α in ischemic aged skeletal muscle. The ischemic soleus of old mice showed a lower mitochondrial respiratory control ratio in POD 21 compared to young mice, which was recovered in MitoTEMPO-treated old mice. Scavenging of mitochondrial superoxide attenuated mitochondrial DNA damage and preserved the mitochondrial respiration, in addition to suppression of the expression of p53 and preservation of the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in ischemic skeletal muscles with aging. Resolution of excessive mitochondrial superoxide could be an effective therapy to recover blood flow of skeletal muscle during ischemia in senescence.

Essential Role of IL-12 in Angiogenesis in Type 2 Diabetes

Recently, IL-12 emerged as a critical player in type 2 diabetes complications. We previously reported that ischemia-induced angiogenesis is compromised in type 2 diabetic mice. In this study, we determined that IL-12 disruption rescued angiogenesis and arteriogenesis in type 2 diabetic mice. To induce type 2 diabetes, wild-type (WT), p40IL-12-/- (p40-/-), and p35IL-12-/- (p35-/-) mice were fed a high-fat diet (HFD) for 12 weeks. Body weight, glucose test tolerance, and insulin test tolerance were assessed. After 12 weeks of an HFD, the femoral artery was ligated and blood flow recovery was measured every week for 4 weeks. WT, p40-/-, and p35-/- mice fed an HFD become obese after 12 weeks and exhibit glucose intolerance and insulin resistance. Blood flow recovery was fully restored in 2 to 3 weeks after femoral artery ligation in all groups of mice fed a normal diet. However, after 12 weeks of an HFD, blood flow recovery was compromised in WT mice, whereas it was fully recovered in p40-/- and p35-/- mice. The mechanism of blood flow recovery involves an increase in capillary/arteriole density, endothelial nitric oxide synthase/Akt/vascular endothelial growth factor receptor 2 signaling, and a reduction in oxidative stress and inflammation. The disruption of IL-12 promotes angiogenesis and increases blood flow recovery in obese type 2 diabetic mice by an endothelial nitric oxide synthase/Akt/vascular endothelial growth factor receptor 2/oxidative stress-inflammation-dependent mechanism.

Acute and chronic effects of exercise on mRNA expression in the skeletal muscle of two mouse models of peripheral artery disease.

Endurance exercise improves walking performance in patients with peripheral artery disease (PAD), which is characterized by skeletal muscle dysfunction caused by lower extremity ischemia. Although transcriptional analyses of exercise-induced changes in normal animals and healthy volunteers have been reported, no detailed study has explored exercise-induced alterations in gene expression in PAD animal models. Here, we determined the acute and chronic effects of exercise on mRNA expression in the skeletal muscles of two mouse models of PAD. Three particular gene categories were investigated: known exercise-responsive genes (Pgc1a, Il6, Nr4a1, Nr4a2, and Nr4a3); myogenic and muscle regeneration-related genes (Myf5, Myogenin, Myomaker, and Myh3); and Gpr56 and its ligand Col3a1. PAD was induced by bilateral femoral artery ligation in normal C57BL/6 and diabetic KK-Ay mice. From 1 week after surgery, repetitive twice-weekly 30-min treadmill endurance exercise sessions were applied. Altered mRNA expression in the soleus muscles was measured in both the acute and chronic phases. In the acute phase, transcript levels of exercise-inducible genes showed significant increases in both C57BL/6 and diabetic KK-Ay PAD mice; levels of regeneration-related genes showed little alteration, and those of Gpr56 increased immediately and significantly after exercise in both models. In the chronic phase, transcript levels of Pgc1a, Myf5, Myogenin, Myomaker, Myh3, Gpr56, and Col3a1 were upregulated significantly in sedentary C57BL/6 PAD mice compared with that in sham-operated mice. Exercise training inhibited the upregulation of Col3a1, Myf5, and Myogenin significantly. In KK-Ay PAD mice, only Gpr56 mRNA levels increased significantly compared with those in sham-operated mice. RNA sequence analysis revealed 33 and 166 differentially upregulated, and 363 and 99 downregulated, genes after exercise training in C57BL/6 PAD and KK-Ay PAD mice, respectively. In summary, we detected significant alterations of skeletal muscle genes after exercise in PAD mouse models and characterized their expression patterns.

Abstract 23: Exosome Inhibition Improved Blood Perfusion in Ischemic Hindlimb of db/db Diabetic Mice

Background: Critical limb ischemia (CLI), a life-threatening condition characterized by pain at rest and tissue loss with ulcer and gangrene, imposes a major public healthy burden, resulting in high mortality and disability. The occurrence of CLI in patients with diabetes mellitus is very frequent. However, the effective therapy for CLI in diabetic patients is absent. Recent studies demonstrated that exosome from diabetic animals/cells has detrimental effects on the post-injury cardiovascular repair. Here, we tested the hypotheses that exosome inhibition in vivo improves blood flow recovery and protects skeletal muscle in ischemic hindlimbs of diabetic db/db mice following surgical ischemia. Methods and Results: Exosomes were isolated from bone-marrow derived progenitor cells or plasma in non-diabetic db/+ and diabetic db/db mice by ultracentrifugation. Diabetic exosome (5 ug/ml) inhibited tube formation of human cardiac microvascular endothelial cells. Unilateral hindlimb ischemia surgery was conducted by ligation of left femoral artery in 12-week old, male db/+ and db/db mice. Exosome inhibitor GW4869 (2 μg/g body weight) was given by intraperitoneal injection every other day for 4 weeks starting from one week before the HLI surgery. HLI mice injected with vehicle served as controls. Mice were divided into four groups: 1) db/+ + vehicle; 2) db/db+ vehicle; 3) db/+ GW4869; 4) db/db + GW4869. GW4869 decreased necrosis and loss of toe/toenail, improved blood flow, enhanced capillary/arterial density, skeletal muscle architecture and cell survival in ischemic hindlimb of diabetic db/db mice 21 days post-ligation. Conclusions: Although preliminary, our experiments suggest that therapeutic targeting of dysfunctional exosome secretion could represent a new avenue for the prevention and treatment of ischemic injury in diabetic patients.

Abstract 370: Wt1 Influences Regenerative Angiogenic Function of the Vascular Endothelium

Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis (new blood vessel formation from existing vasculature) fails to consistently induce sufficient reperfusion to facilitate repair, a targeted approach to manipulating angiogenesis is clinically desirable. The Wilms’ tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that Wt1 influences the angiogenic function of endothelial cells. Wt1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from Wt1-GFP reporter mice. Inducible deletion of Wt-1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p&lt;0.05 relative to Cre and vehicle controls, n=5-8). In vivo , Wt-1 expression was evident in vascular endothelial cells and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia. Less expression was detected by day 28. VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density (histology, immunohistochemistry) or expression of angiogenic factors (qRT-PCR) in sponges inserted beneath the skin of mice (20 days). To further understand the role of Wt1 in angiogenesis, transcriptomic analysis was performed for RNA expression in Wt1 + and Wt1 - cells isolated (FACs) from sponges 7 and 21 days after implantation in Wt1-GFP mice. Wt1 + cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including vasculature development (D7: p=10 -7.43 , D21: p=10 -3.47 , n=3) and angiogenesis (D7: p=10 -3.90 , D21: p=10 -4.74 , n=3). These results show that deletion of endothelial Wt-1 delays, rather than permanently inhibiting, angiogenesis: suggesting a more complex mechanism of action than reported previously. Transcriptomic data suggest that expression in endothelial cells (and other cells in the endothelial lineage) contributes to tissue repair in ischaemia. This work has improved our understanding of the regulatory role of Wt1 in angiogenesis.

Bradykinin does not acutely sensitize the reflex pressor response during hindlimb skeletal muscle stretch in decerebrate rats.

Hindlimb skeletal muscle stretch (i.e., selective activation of the muscle mechanoreflex) in decerebrate rats evokes reflex increases in blood pressure and sympathetic nerve activity. Bradykinin has been found to sensitize mechanogated channels through a bradykinin B2 receptor-dependent mechanism. Moreover, bradykinin B2 receptor expression on sensory neurons is increased following chronic femoral artery ligation in the rat (a model of simulated peripheral artery disease). We tested the hypothesis that injection of bradykinin into the arterial supply of a hindlimb in decerebrate, unanesthetized rats would acutely augment (i.e., sensitize) the increase in blood pressure and renal sympathetic nerve activity during hindlimb muscle stretch to a greater extent in rats with a ligated femoral artery than in rats with a freely perfused femoral artery. The pressor response during static hindlimb muscle stretch was compared before and after hindlimb arterial injection of 0.5 µg of bradykinin. Injection of bradykinin increased blood pressure to a greater extent in "ligated" (n = 10) than "freely perfused" (n = 10) rats. The increase in blood pressure during hindlimb muscle stretch, however, was not different before vs. after bradykinin injection in freely perfused (14 ± 2 and 15 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.62) or ligated (15 ± 3 and 14 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.80) rats. Likewise, the increase in renal sympathetic nerve activity during stretch was not different before vs. after bradykinin injection in either group of rats. We conclude that bradykinin did not acutely sensitize the pressor response during hindlimb skeletal muscle stretch in freely perfused or ligated decerebrate rats.

Aucubin promotes angiogenesis via estrogen receptor beta in a mouse model of hindlimb ischemia

Aucubin (AU) is an iridoid glycoside that has been shown to display estrogenic properties and has various pharmacological effects. Herein, we described the angiogenic properties of AU. In the study, hindlimb ischemia was induced by ligation of femoral artery on the right leg of ovariectomized mice. AU treatment significantly accelerated perfusion recovery and reduced tissue injury in mice muscle. Quantification of CD31-positive vessels in hindlimb muscles provided evidences that AU promoted angiogenesis in peripheral ischemia. In addition, results from quantitative PCR and western blot suggested AU induced angiogenesis via vascular endothelial cell growth factor (VEGF)/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway. More interestingly, AU’s angiogenic effects could be completely abolished in estrogen receptor beta (ERβ) knockout mice. In conclusion, the underlying mechanisms were elucidated that AU produced pro-angiogenic effects through ERβ-mediated VEGF signaling pathways. These results expand knowledge about the beneficial effects of AU in angiogenesis and blood flow recovery. It might provide insight into the ERβ regulating neovascularisation in hindlimb ischemia and identify AU as a potent new compound used for the treatment of peripheral vascular disease.

Ager Deletion Enhances Ischemic Muscle Inflammation, Angiogenesis, and Blood Flow Recovery in Diabetic Mice.

Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block angiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated angiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1 (early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, angiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. These findings indicate that RAGE attenuates adaptive inflammation in hindlimb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic peripheral vascular disease.

Abstract 119: Protective Effects of Chloroquine on Ischemic and Nutrient Depleted Muscle are Mediated by HMGB1 and Caspase-1

Introduction: Millions of Americans are at risk for amputation from severe peripheral arterial disease (PAD) when surgery is not possible. Pro-regenerative and angiogenic agents may improve outcome in that setting. Chloroquine (CQ) promotes wound healing in scleroderma but has not been tested in PAD. CQ promotes healing of ischemic muscle, increases muscle high mobility group box 1 (HMGB1), an inflammatory, pro-angiogenic protein, and activates caspase-1 in myoblasts. We hypothesize that HMGB1 mediates protective effects of CQ and is regulated by caspase-1 in muscle. Controlled rather than indiscriminate release of HMGB1 from damaged muscle may be protective during ischemia. Methods: C2C12 myoblasts in low serum were treated with CQ (0-50μM) ± Ac-YVAD-cmk (10 μg/ml), a caspase-1 inhibitor. HMGB1 release in supernatants was measured using ELISA. Cytotoxicity was assessed by comparing spontaneous lactate dehydrogenase (LDH) activity in culture media from control, treated and maximally lysed cells. CQ (50μg/ml) or placebo treated wild-type and inducible HMGB1 knockout (iHMGB1KO) mice underwent unilateral femoral artery ligation (FAL). Laser Doppler perfusion imaging (LDPI) before and 1,7,14 and 21d after FAL was reported as % improvement over time. ANOVA was used to assess statistical significance among groups. Results: CQ (5-10uM) attenuated spontaneous LDH leak after 12h from serum-depleted myoblasts (p &lt;0.01, N=3), and modestly increased HMGB1 release (p &lt;0.001, N=3). Ac-YVAD-cmk reversed the cytoprotective effects of CQ, significantly raising both LDH activity to 55% of maximal activity and HMGB1 in the supernatant. Compared to d1 post FAL, CQ improved perfusion recovery in WT mice by 300-800% over 21 days (p&lt;0.03, N=7/group), but not in iHMGB1KO mice. Conclusion: We present the novel finding that in nutrient-depleted myoblasts, caspase-1 mediates the survival benefits of CQ and regulates HMGB1 release. In turn, HMGB1 is critical for CQ’s beneficial effects on perfusion after FAL, another stress condition. Regulated HMGB1 release may be immunomodulatory, regenerative and modifiable with drugs like CQ. Altering survival and inflammatory pathways through CQ may present a novel therapeutic strategy in PAD.

Abstract 475: Novel Gene Therapy Facilitates Critical Limb Ischemia Reperfusion in Mouse Hindlimb Gangrene Model

Lack of a reliable animal hindlimb gangrene model limits molecular investigation of critical limb ischemia. We sought to develop a mouse hindlimb gangrene model, used to assess the efficacy of novel gene therapy. We hypothesized that priming ischemic hindlimb tissue with E-selectin/adeno-associated virus (AAV) enhances therapeutic angiogenesis and attenuates gangrene. We tested two methods to induce hindlimb gangrene. First, FVB mice underwent femoral artery ligation (FAL) to achieve critical limb ischemia. Second, FVB underwent combined FAL and administration of NG-nitro-L-arginine methyl ester (L-NAME), nitric oxide synthase inhibitor, which further reduces hindlimb perfusion. Prior to FAL and L-NAME use, gangrene-induced mice were intramuscularly administered E-selectin/AAV (treatment) or LacZ/AAV (control) to the hindlimb. Gangrene was assessed with a standardized ischemia score ranging from 0 (no gangrene) to 11 (forefoot gangrene), recorded on postoperative day (POD)’s 2,7,14. Hindlimb reperfusion using Laser doppler imaging was quantified by mean perfusion of ligated:non-ligated limb on same POD’s. Live animal Dil perfusion plus laser scanning confocal microscopy quantified limb neovascularization. Most FVB did not develop gangrene with FAL-only (n=2/8, 25% gangrene incidence). Combined FAL and L-NAME consistently induced hindlimb gangrene (n=14/14, 100% gangrene incidence). Laser doppler imaging score on POD 7 for E-selectin/AAV (n=7) and LacZ/AAV (n=7) was 0.41 vs 0.27 (P=0.071) and on POD 14 was 0.54 vs 0.29 (P=0.017). Dil perfused ligated hindlimb in E-selectin/AAV and LacZ/AAV revealed significantly higher mean neovascularization intensity score of 44 vs 21 (P=0.037). Dil perfused non-ligated limb in respective mice demonstrated mean intensity score of 50 vs 25 (P=0.006). Mean limb ischemia score on POD 2,7,14 for E-selectin/AAV and LacZ/AAV was 1.9, 2.9, 3.7 vs 2.7, 3.9, 5.3 (P=0.104). We developed a highly reliable mouse hindlimb gangrene model where E-selectin-based novel gene therapy improved limb reperfusion and neovascuclarization in critical limb ischemia. This hindlimb gangrene model can be used to further understand Redox pathways contributing to gangrene, facilitating future translational studies.

Endothelial deletion of mTORC1 protects against hindlimb ischemia in diabetic mice via activation of autophagy, attenuation of oxidative stress and alleviation of inflammation

Peripheral arterial disease (PAD) complicated with diabetes mellitus (DM) still remains a thorny issue due to lack of effective strategies. Our previous study has demonstrated that inhibition of mTORC1 protected adipose-derived stromal cells from hindlimb ischemic injury in PAD mice. However, whether inhibition of mTORC1 could protect against PAD in diabetes mellitus and the underlying mechanisms remained elusive. In this study, we employed endothelial-specific raptor (an essential component of the mTORC1 signaling complex) knockout (KO) mice (Tie2-mTORC1ko) to investigate whether and how mTORC1 downregulation could alleviate hindlimb ischemic injury in diabetic mice. Tie2-mTORC1ko mice and their wild-type littermates were intraperitoneally injected with streptozocin to induce type 1 diabetic model, after which the hyperglycemic mice were randomly allocated to sham operation or PAD operation (femoral artery ligation). The restoration of hindlimb blood perfusion and recovery of limb functions were improved in diabetic Tie2-mTORC1ko PAD mice with significant improvements of autophagy, angiogenesis and vascular integrity as well as attenuation of apoptosis, inflammation and oxidative stress. In vitro, high glucose combining with hypoxia/serum deprivation treatment (HG+H/SD) significantly triggered apoptosis, reactive oxygen species generation and inflammation while inhibited autophagy and tube formation in HUVECs. The effect could be accentuated and attenuated by mTORC1 over-expression (TSC2 siRNA) and mTORC1 silencing (raptor siRNA), respectively. Moreover, autophagy inhibitor 3-MA could simulate the effects of TSC2 siRNA while autophagy inducer rapamycin could mimic the effects of raptor siRNA, suggesting that the beneficial effects of mTORC1 deletion were associated with autophagy induction. In conclusion, our present study demonstrates that endothelial mTORC1 deletion protects against hindlimb ischemic injury in diabetic mice possibly via activation of autophagy, attenuation of oxidative stress and alleviation of inflammation. Therapeutics targeting mTORC1 may therefore represents a promising strategy to rescue limb ischemia in diabetes mellitus.

Diabetes Impairs the Ability of Skeletal Muscle Pericytes to Augment Postischemic Neovascularization in Mice

Peripheral artery disease is an atherosclerotic disease that causes limb ischemia and has few effective treatments. Stem cell therapy is a promising treatment option, but concomitant diabetes may limit its effectiveness. PURPOSE: To evaluate the therapeutic potential of skeletal muscle pericytes to augment postischemic neovascularization following the induction of limb ischemia in wild type (WT) and type 2 diabetic (T2DM) mice. METHODS: Pericytes were isolated via fluorescence activated cell sorting for CD45-CD34-CD146+ cells, and pericyte phenotype was confirmed via surface marker expression, gene expression, and in vitro differentiation potential. WT C57BL/6 (n=10) and db/db T2DM (n=8) mice underwent unilateral femoral artery ligation to induce limb ischemia. 24 hrs post-ligation, pericytes or vehicle control were transplanted into the muscles of the ischemic hindlimbs. Postischemic neovascularization was assessed via foot blood flow at pre-surgery, post-surgery, and postoperative days 3, 7, 14, 21, and 28 using laser Doppler perfusion imaging. Differences in gene expression were determined using t-tests; differences in blood flow were determined using linear mixed models. RESULTS: CD45-CD34-CD146+ pericytes were positive for mesenchymal stem cell markers CD90 (74%) and CD105 (65%) and weakly positive for the pericyte marker PDGFRβ (42%) and the endothelial cell marker CD144 (36%). Pericytes transdifferentiated into skeletal myocytes, adipocytes, osteocytes, and endothelial cells. Pericytes had significantly (p<0.05) upregulated Sca1 (4.0-fold) gene expression, downregulated CD31 (0.2-fold) gene expression, and no difference in MyoD (1.0-fold), Pax3 (1.3-fold), or Pax7 (1.0-fold) expression. Blood flow recovery in WT mice was significantly higher after pericyte transplantation than after vehicle control (p=0.03; 81.1±6% vs. 64.7±9% at postoperative day 28). Blood flow recovery in T2DM mice after pericyte transplantation was not different than after vehicle control (p=0.44; 50.1±4% vs. 53.5±4% at postoperative day 28). CONCLUSION: T2DM negates the capacity of pericytes to augment neovascularization after limb ischemia. Further study is required to determine the mechanism by which T2DM impairs this pericyte function. Support: AAUW American Dissertation Fellowship

BAG3 (Bcl-2-Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy.

Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1, that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6-Lsq1-3). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein- (n=29) expressing animals. BAG3Ile81, but not BAG3Met81, improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus-BAG3Ile81 (n=9), but not BAG3Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3Met81, BAG3Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Beneficial Effects of Sildenafil on Tissue Perfusion and Inflammation in a Murine Model of Limb Ischemia and Atherosclerosis.

Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, has endothelium protective and angiogenic effects. To test if sildenafil improves tissue perfusion and neovascularization and downregulates proinflammatory molecules following limb ischemia. 30 ApoE-/- male mice, bred with cholesterol rich diet for 4 weeks, were anesthetized and underwent unilateral hind-limb ischemia with ligation of the left femoral artery. Mice were randomized in 2 groups: sildenafil (1 mg/Kg for 7 days intraperitoneally, i.p.) or normal saline (0.4 ml for 7 days, i.p.). Bilateral hind-limb perfusion was estimated by laser Doppler imaging after surgery on days 0, 7 and 28. Sildenafil significantly reduced at day 28 compared with day 0 levels of soluble intracellular adhesion molecule-1(sICAM-1) [2.24(1.81-2.41) vs. 1.29(0.87-1.45) ng/ml, p=0,01], soluble E-selectin (sE-Selectin) [5.52 (3.67-6.14) vs 1.71 (1.42-2.86) ng/ml, p=0.02] and tissue plasminogen activator inhibitor- 1 (tPAI-1) [0.13(0.07-0.21) vs 0.08 (0.04-0.10) ng/ml, p=0.01] while normal saline had no effect on the levels of sICAM-1, sE-Selectin and tPAI-1. Treatment with sildenafil was associated with increased perfusion in the ischemic limb compared with controls. Sildenafil exerts significant beneficial effects on tissue perfusion and inflammatory status after limb ischemia, a finding implying neovascularization and potential vascular protective properties of sildenafil.

Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle

Here we investigated whether endothelial colony forming cells (ECFC) and mesenchymal progenitor cells (MPC) form vascular networks and restore blood flow in ischemic skeletal muscle, and whether host myeloid cells play a role. ECFC + MPC, ECFC alone, MPC alone, or vehicle alone were injected into the hind limb ischemic muscle one day after ligation of femoral artery and vein. At day 5, hind limbs injected with ECFC + MPC showed greater blood flow recovery compared with ECFC, MPC, or vehicle. Tail vein injection of human endothelial specific Ulex europaeus agglutinin-I demonstrated an increased number of perfused human vessels in ECFC + MPC compared with ECFC. In vivo bioluminescence imaging showed ECFC persisted for 14 days in ECFC + MPC-injected hind limbs. Flow cytometric analysis of ischemic muscles at day 2 revealed increased myeloid lineage cells in ECFC + MPC-injected muscles compared to vehicle-injected muscles. Neutrophils declined by day 7, while the number of myeloid cells, macrophages, and monocytes did not. Systemic myeloid cell depletion with anti-Gr-1 antibody blocked the improved blood flow observed with ECFC + MPC and reduced ECFC and MPC retention. Our data suggest that ECFC + MPC delivery could be used to reestablish blood flow in ischemic tissues, and this may be enhanced by coordinated recruitment of host myeloid cells.

Chronic Femoral Artery Ligation Exaggerates the Exercise Pressor Reflex but Not Selective Activation of the Muscle Mechanoreflex in Decerebrate Rats

Mechanical and metabolic stimuli arising within contracting skeletal muscles reflexly increase sympathetic nervous system activity, blood pressure, and heart rate. This reflex, termed the exercise pressor reflex, is exaggerated in a rat model of limb ischemia in which a femoral artery is chronically ligated. We recently found that the mechanically‐sensitive component of the exercise pressor reflex contributes to its exaggeration in rats with a ligated femoral artery despite no difference in mechano‐gated channel protein expression in lumbar dorsal root ganglia (DRG) tissue. This suggests that metabolite‐induced sensitization of mechanoreceptors underlies the exaggerated mechanically‐sensitive component of the exercise pressor reflex in ligated rats. This prompted us to seek the physiological evidence in support of the anatomical evidence that ligation does not increase mechano‐gated channel expression in DRG tissue.ObjectiveWe used a within‐rat comparison experimental design to test the hypothesis that the increase in blood pressure, heart rate, and renal sympathetic nerve activity (RSNA) during selective activation of the muscle mechanoreflex (a purely mechanical stimulus) would not be different when evoked from a freely perfused hindlimb compared a ligated hindlimb.MethodsFifteen male Sprague‐Dawley rats had their left femoral artery ligated 72 hours before the experiment. In decerebrate, unanesthetized rats the muscle mechanoreflex was selectively activated by passively stretching the Achilles tendon for 30 s. In five of those fifteen rats, the exercise pressor reflex was evoked by electrically‐stimulating the sciatic nerves for 30 s which produced static hindlimb muscle contractions.ResultsIn 10 rats there were no differences between the peak pressor (freely perfused: 13±2, ligated: 14±3 mmHg, p=0.801), cardioaccelerator (freely perfused: 12±2, ligated: 11±2 bpm, p=0.672), or RSNA (freely perfused: 40±7, ligated: 46±6%, p=0.377) responses evoked by tendon stretch of the freely perfused and ligated hindlimbs. In five additional rats, the exercise pressor reflex was greater when evoked from the contracting ligated hindlimb (28±3 mmHg) compared to the contracting freely perfused hindlimb (21±3 mmHg, p=0.014). In those same five rats, however, in which femoral artery ligation exaggerated the exercise pressor reflex there was no difference in the peak pressor response to tendon stretch between hindlimbs (freely perfused: 16±2, ligated: 15±3 mmHg, p=0.581).ConclusionWe found that the reflex pressor, cardioaccelerator, and RSNA responses to tendon stretch (a purely mechanical stimulus) were not exaggerated by femoral artery ligation, even in rats in which the exercise pressor reflex was confirmed to be exaggerated by ligation. These findings indicate that the ligation‐induced exaggeration of the mechanically‐sensitive component of the exercise pressor reflex reported previously must be due to a metabolite‐induced sensitization of mechano‐gated receptors.Support or Funding InformationDepartmental funds

17 Femoral artery ligation induces rapid arteriogenesis that correlates with recovery of perfusion in the foot

Background and hypothesis Reperfusion following acute hindlimb ischaemia is mediated by both arteriogenesis (collateral growth) and angiogenesis. According to Poiseuille’s Law, the arterial ability to conduct blood is proportional to the 4th order of its diameter. This suggests collateral arteries with a relatively large diameter are better suited to conduct blood than smaller vessels. We hypothesise that arteriogenic remodelling of collateral vessels has a greater role than angiogenesis in restoring blood supply to the ischaemic hindlimb. Methods Acute hindlimb ischaemia was induced in 12-week-old male C57BL/6J mice by unilateral femoral artery ligation. Reperfusion in the foot pad was monitored by laser Doppler immediately before and after femoral artery ligation and subsequently on days 1, 3 and 7 (n=3 for each time point). Vascular resin casts plus optical projection tomography (OPT) were used to illustrate the arterial network before and after ischaemia. Results Blood perfusion in the foot pad dropped by ~90% immediately following ligation but exhibited a 40% recovery over the next 7 days. This restoration of flow correlated with the emergence and growth of collateral arteries in the thigh (upstream of the ligation point). Conclusion A pre-existing collateral circulation provides the residual blood supply after femoral ligation. A rapid increase in the diameter of a small number of collateral arteries appeared to be the major mechanism for acute restoration of blood supply to the ischaemic lower leg and foot pad. Future work will use histology and immunohistochemistry to investigate the role of angiogenesis in reperfusion following femoral ligation.

Mechanoadaptation, arteriogenesis, and vascular reactivity in male and female C57Bl/6 mice

Women have a higher incidence of peripheral arterial occlusive disease (PAOD) and a greater likelihood for developing critical limb ischemia. The importance of the collateral circulation in maintaining tissue blood flow following arterial occlusion suggests that this sexually‐dimorphic response may be due to sex differences in collateral development and function. In experimental animals, females exhibit impaired regional recovery (i.e. limb pressure or resistance) that may not may not require estrogen deficiency. Although the collateral circulation is the primary site of blood flow resistance following arterial occlusion and the likely explanation of any impairment in hindlimb recovery, neither of these conflicting reports directly evaluated collateral enlargement or function. Therefore, we tested the hypothesis that collateral enlargement and reactivity were impaired in female mice.All experiments were performed in C57Bl/6 mice at day‐7 or day‐28 following femoral artery ligation (FAL), between the epigastric/profunda and popliteal branches. In the stem to the collateral circulation (i.e. the profunda femoris), enlargement does not involve cellular proliferation, so we evaluated mecahnoadaptive outward remodeling by measuring vascular smooth muscle cell length and overlap with immunofluorescence and confocal microscopy. At day‐7 following FAL, vascular smooth muscle cell (VSMC) length increased 25% in both male and female mice. Similarly, VSMC overlap decreased by 30% in both male and female mice‐indicating roughly equivalent mechanoadaptive outward remodeling. At the midzone of the gracilis anterior collateral, inner and outer diameter increased 100% in both males and females, while wall thinkness increased over 200%, indicating similar levels of hypertrophic outward remodeling in both sexes. Given the lack of difference in enlargement between male and female collaterals, we next evaluated reactivity, to determine if female mice exhibit impaired collateral function. As expected, reactivity was substantially impaired at day‐7 following FAL, with collateral midzone diameter increasing 15% during functional vasodilation, as compared to nearly 60% increases in sham‐operated contralateral collaterals; responses were not different between males and females. At day‐28 following FAL, collateral midzone reactivity in males was restored, and no longer different from the contralateral control, with both increasing 75% during functional vasodilation. Surprisingly, collateral midzone reactivity was enhanced in females, as compared to both males and the contralateral control, increasing 150% during functional vasodilation.If these data are representative of all collateral vessels, they suggest that impairments in flow recovery following arterial occlusion in female mice with intact ovaries are likely due to dysfunction in the distal microvasculature. Since female mice exhibit more impaired vasodilation during hypercholesterolemia, we will evaluate the collateral enlargement and function in a more clinically‐relevant animal model with vascular disease risk factors in future animal studies aimed at understanding the worse prognosis of females patients with PAOD.