Femoral Artery Ligation Model Research Articles

Growth factor free, peptide-functionalized gelatin hydrogel promotes arteriogenesis and attenuates tissue damage in a murine model of critical limb ischemia

Critical limb ischemia (CLI) occurs when blood flow is restricted through the arteries, resulting in ulcers, necrosis, and chronic wounds in the downstream extremities. The development of collateral arterioles (i.e. arteriogenesis), either by remodeling of pre-existing vascular networks or de novo growth of new vessels, can prevent or reverse ischemic damage, but it remains challenging to stimulate collateral arteriole development in a therapeutic context. Here, we show that a gelatin-based hydrogel, devoid of growth factors or encapsulated cells, promotes arteriogenesis and attenuates tissue damage in a murine CLI model. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins. Mechanistically, these “GelCad” hydrogels promote arteriogenesis by recruiting smooth muscle cells to vessel structures in both ex vivo and in vivo assays. In a murine femoral artery ligation model of CLI, delivery of in situ crosslinking GelCad hydrogels was sufficient to restore limb perfusion and maintain tissue health for 14 days, whereas mice treated with gelatin hydrogels had extensive necrosis and autoamputated within 7 days. A small cohort of mice receiving the GelCad hydrogels were aged out to 5 months and exhibited no decline in tissue quality, indicating durability of the collateral arteriole networks. Overall, given the simplicity and off-the-shelf format of the GelCad hydrogel platform, we suggest it could have utility for CLI treatment and potentially other indications that would benefit from arteriole development.

A biomaterial-based therapy for lower limb ischemia using Sr/Si bioactive hydrogel that inhibits skeletal muscle necrosis and enhances angiogenesis.

Muscle necrosis and angiogenesis are two major challenges in the treatment of lower-limb ischemic diseases. In this study, a triple-functional Sr/Si-containing bioceramic/alginate composite hydrogel with simultaneous bioactivity in enhancing angiogenesis, regulating inflammation, and inhibiting muscle necrosis was designed to treat lower-limb ischemic diseases. In particular, sodium alginate, calcium silicate and strontium carbonate were used to prepare injectable hydrogels, which was gelled within 10min. More importantly, this composite hydrogel sustainedly releases bioactive Sr2+ and SiO3 2- ions within 28 days. The biological activity of the bioactive ions released from the hydrogels was verified on HUVECs, SMCs, C2C12 and Raw 264.7cells in vitro, and the therapeutic effect of the hydrogel was confirmed using C57BL/6 mouse model of femoral artery ligation in vivo. The results showed that the composite hydrogel stimulated angiogenesis, developed new collateral capillaries, and re-established the blood supply. In addition, the bioactive hydrogel directly promoted the expression of muscle-regulating factors (MyoG and MyoD) to protect skeletal muscle from necrosis, inhibited M1 polarization, and promoted M2 polarization of macrophages to reduce inflammation, thereby protecting skeletal muscle cells and indirectly promoting vascularization. Our results indicate that these bioceramic/alginate composite bioactive hydrogels are effective biomaterials for treating hindlimb ischemia and suggest that biomaterial-based approaches may have remarkable potential in treating ischemic diseases.

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)

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.

Exaggerated exercise pressor reflex in a mouse model of femoral artery ligation

The exercise pressor reflex (EPR) is a reflexive mechanism in which blood pressure and heart rate increase in response to skeletal muscle contraction. These reflexive increases are caused by both mechanical and metabolic stimulation of group III and IV afferent neurons innervating in contracting skeletal muscle. Patients with peripheral artery disease (PAD) have an exaggerated EPR which contributes to exercise intolerance and morbidity and mortality. Previous studies in our laboratory have demonstrated that the EPR is overactive in a rat model of heart failure similar to it is in human heart failure patients. We determined that this abnormality was initiated by a blunting of the metabolic component but maintained by the mechanosensitive component of the reflex. Recently, we developed a mouse model of EPR, and have validated that the mouse model is similar to our rat model. In this model, studied the EPR in a model of peripheral artery disease (PAD). Our current results demonstrated that the blood pressure increase induced by passive stretch was exaggerated in mice 72 hours following femoral artery ligation. This exaggerated rise in blood pressure was not affected by SB-705498 (a TRPv1 receptor inhibitor) but it was nearly abolished by Gadolinium (a mechanical sensitive group III afferent neurons inhibitor) and it was partially reduced by GsMTx-4 (a selective inhibitor of the Piezo2 receptor). These results indicate that the exaggerated EPR in a murine model of PAD is mediated by mechanically sensitive group III neurons, which is mediated, in part, by Piezo2 receptors.

HIF-1α inhibition alleviates the exaggerated exercise pressor reflex in rats with peripheral artery disease induced by femoral artery occlusion.

Hypoxia‐inducible factor 1α (HIF‐1α) is a transcription factor mediating adaptive responses to hypoxia and ischemia. Our previous work showed that HIF‐1α is increased in muscle sensory nerves of rats with peripheral artery disease (PAD) induced by femoral artery occlusion. The present study was further to examine the role played by HIF‐1α in regulating the response of arterial blood pressure (BP) to the activation of muscle afferent nerve during static muscle contraction in rats with femoral artery occlusion. A rat model of femoral artery ligation was used to study PAD in this study. Western blot analysis was employed to examine the protein levels of HIF‐1α in the dorsal root ganglion (DRG) tissues. BAY87, a synthesized compound with the characteristics of highly potent and specific suppressive effects on expression and activity of HIF‐1α, was given into the arterial blood supply of the ischemic hindlimb muscles before the exercise pressor reflex was evoked by static muscle contraction. First, HIF‐1α was increased in the DRG of occluded limbs (optical density: 0.89 ± 0.13 in control versus 1.5 ± 0.05 in occlusion; p < 0.05, n = 6 in each group). Arterial injection of BAY87 (0.2 mg/kg) then inhibited expression of HIF‐1α in the DRG of occluded limbs 3 hr following its injection (optical density: 1.02 ± 0.09 in occluded limbs with BAY87 versus 1.06 ± 0.1 in control limbs; p > 0.05, n = 5 in each group). In addition, muscle contraction evoked a greater increase in BP in occluded rats. BAY87 attenuated the enhanced BP response in occluded rats to a greater degree than in control rats. Our data suggest that the inhibition of HIF‐1α alleviates the exaggeration of the exercise pressor reflex in rats under ischemic circumstances of the hindlimbs in PAD induced by femoral artery occlusion.

591-P: Macrophage IL-1beta–Dependent VEGF-A Expression Is Required for Inflammatory Arteriogenesis and Wound Healing in Type 2 Diabetes

Impaired wound healing in patients with diabetes mellitus (DM) is a major source of morbidity and mortality. Current treatments for diabetic wounds are modestly effective at promoting healing. Recently, we identified an important role for inflammatory macrophage VEGF-A production in consequent angiogenesis/arteriogenesis required for adequate wound healing. We sought to understand the impact of diabetes on inflammatory angiogenesis and consequent healing. We hypothesized that diabetes mellitus (DM) results in reduced inflammatory macrophage VEGF-A expression with consequent impairments in angiogenesis-dependent wound healing. Mice with experimental diabetes demonstrated profound delays both in wound healing using a dermal punch biopsy model and in perfusion recovery using the femoral artery ligation model of hind limb ischemia. VEGF-A expression is highly upregulated in “classic inflammatory” macrophages relative to “alternatively activated” or wound healing macrophages. This is supported by a strong association between IL-1β and VEGF-A expression. In order to understand whether this relationship between IL-1β and VEGF-A was causal, we generated a novel mouse with a LoxP flanked IL-1β allele to create an inducible macrophage deletion of IL-1β. Myeloid IL-β deletion resulted in severely impaired macrophage VEGF-A expression and consequent decreases in angiogenesis and wound healing, consistent with the experimental model of DM. Moreover, macrophages from diabetic mice demonstrated reduced expression of IL-1R signaling complex components along with consequent reductions in VEGF-A expression. Defining inflammatory macrophages as key, early drivers of angiogenesis supports a paradigm shift away from inflammation-suppression for adequate wound healing, allowing for macrophage reprograming strategies that promote appropriate inflammation-dependent healing responses. Disclosure C.S. Mantsounga: None. Funding National Institutes Health (1R01HL139795)

Histopathological significance of microRNA-210 expression in acute peripheral ischemia in a murine femoral artery ligation model.

Under hypoxic conditions, microRNA-210 is upregulated and plays multiple physiological roles including in cell growth arrest, stem cell survival, repression of mitochondrial respiration, angiogenesis, and arrest of DNA repair. In this study, we investigated the histopathological expression of microRNA-210 under hypoxic conditions using a femoral artery ligation model established in C57BL/6J mice to determine the pathological significance of microRNA-210. Following femoral artery ligation, ischemia was represented by decreased blood flow compared to the control, in which a sham operation was performed. On histopathology, degeneration/necrosis of the muscle fibers, inflammatory cell infiltration, and regeneration of the muscle fibers were sequentially observed from 3 h to 3 d after ligation of the artery. The degree of these effects was more severe in the area in which type I muscular fibers are dominant. The histological expression of hypoxia-inducible factor 1α, a well-known biomarker of hypoxia, and microRNA-210 was observed in a few necrotic muscle fibers, macrophages, and myoblasts, a distribution consistent with the histopathological lesions, and their signal increased over time. The expression of microRNA-210 in macrophages and myoblasts under ischemia might be indicative of a significant role in the recovery from ischemic lesions. In addition, the in situ hybridization of microRNA-210 could potentially be used for the detection of hypoxia as a histological marker in addition to the immunohistochemistry of hypoxia-inducible factor 1α.

Smooth muscle SIRT1 reprograms endothelial cells to suppress angiogenesis after ischemia

Objective: Vascular smooth muscle cells (VSMCs) undergo the phenotypic changes from contractile to synthetic state during vascular remodeling after ischemia. SIRT1 protects against stress-induced vascular remodeling via maintaining VSMC differentiated phenotype. However, the effect of smooth muscle SIRT1 on the functions of endothelial cells (ECs) has not been well clarified. Here, we explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms.Methods: We performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (SIRT1-Tg) and knockout (KO) mice. Angiogenesis was assessed in in vivo by quantification of the total number of capillaries, wound healing and matrigel plug assays, and in vitro ECs by tube formation, proliferation and migration assays. The interaction of HIF1α with circRNA was examined by using RNA immunoprecipitation, RNA pull-down and in situ hybridization assays.Results: The blood flow recovery was significantly attenuated in SIRT1-Tg mice, and markedly improved in SIRT1-Tg mice treated with SIRT1 inhibitor EX527 and in SIRT1-KO mice. The density of capillaries significantly decreased in the ischemic gastrocnemius of SIRT1-Tg mice compared with SIRT1-KO and WT mice, with reduced expression of VEGFA, which resulted in decreased number of arterioles. We identified that the phenotypic switching of SIRT1-Tg VSMCs was attenuated in response to hypoxia, with high levels of contractile proteins and reduced expression of the synthetic markers and NG2, compared with SIRT1-KO and WT VSMCs. Mechanistically, SIRT1-Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions. Meantime, the high cZFP609 expression was observed in the plasma of the patients with atherosclerotic or diabetic lower extremity peripheral artery disease, associated with reduced ankle-brachial index. Knockdown of cZFP609 improved blood flow recovery after hindlimb ischemia in SIRT1-Tg mice.Conclusions: Our findings demonstrate that SIRT1 may impair the plasticity of VSMCs. cZFP609 mediates VSMCs to reprogram endothelial functions, and serves as a valuable indicator to assess the prognosis and clinical outcomes of ischemic diseases.

Mechanism of angiogenesis promotion with Shexiang Baoxin Pills by regulating function and signaling pathway of endothelial cells through macrophages

Background and aims“Shexiang Baoxin Pill” (SBP), a commonly used traditional Chinese medicine, has been used to treat angina, myocardial infarction and coronary heart disease in China for thirty years. SBP has been proven to promote angiogenesis in a rat model of myocardial infarction (MI). The aim of the present study was to determine the pro-angiogenic effects and mechanism of SBP during inflammation or ischemic pathological conditions and elucidate its regulatory effects on endothelial cell function and signaling pathways mediated by macrophages. MethodsWe used a polyvinyl alcohol (PVA) sponge implantation mouse model as an inflammatory angiogenesis model and utilized a mouse femoral artery ligation model as a hind limb ischemia model. We also performed cell proliferation, cell migration and tubule formation in vitro experiments to assess the effects of SBP on endothelial cell function and signaling pathways by stimulating macrophage activity. ResultsThe in vitro experiment results showed that SBP could significantly increase the expression of mRNAs and proteins associated with angiogenesis in endothelial cells by activating macrophages to release pro-angiogenic factors such as Vegf-a. Activation of macrophages by SBP eventually led to endothelial cell proliferation, migration and tubule formation and increased the expression of p-Akt and p-Erk1/2 proteins in the downstream PI3K/Akt and MAPK/Erk1/2 signaling pathways related to angiogenesis, respectively. The in vivo experiment results indicated that SBP had angiogenesis effects in both inflammatory and ischemic angiogenesis models with dose- and time-dependent effects. ConclusionShexiang Baoxin Pills can promote angiogenesis by activating macrophages to regulate endothelial cell function and signal transduction pathways.

Investigation of the mechanisms of cyclooxygenase-mediated mechanoreflex sensitization in a rat model of simulated peripheral artery disease.

Mechanical and metabolic stimuli within contracting skeletal muscles reflexly increase sympathetic nervous system activity and blood pressure. That reflex, termed the exercise pressor reflex, is exaggerated in patients with peripheral artery disease (PAD) and in a rat PAD model with a chronically ligated femoral artery. The cyclooxygenase (COX) pathway contributes to the exaggerated pressor response during rhythmic skeletal muscle contractions in patients with PAD, but the specific mechanism(s) of the COX-mediated exaggeration are not known. In decerebrate, unanesthetized rats with a chronically ligated femoral artery ("ligated" rats), we hypothesized that hindlimb arterial injection of the COX inhibitor indomethacin would reduce the pressor response during 1-Hz dynamic hindlimb skeletal muscle stretch; a model of the activation of the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex). In ligated rats (n = 7), indomethacin reduced the pressor response during stretch (control: 30 ± 4; indomethacin: 12 ± 3 mmHg; P < 0.01), whereas there was no effect in rats with "freely perfused" femoral arteries (n = 6, control: 18 ± 5; indomethacin: 17 ± 5 mmHg; P = 0.87). In ligated rats (n = 4), systemic indomethacin injection had no effect on the pressor response during stretch. Femoral artery ligation had no effect on skeletal muscle COX protein expression or activity or concentration of the COX metabolite prostaglandin E2. Conversely, femoral artery ligation increased expression of the COX metabolite receptors endoperoxide 4 and thromboxane A2-R in dorsal root ganglia tissue. We conclude that, in ligated rats, the COX pathway sensitizes the peripheral endings of mechanoreflex afferents, which occurs principally as a result of increased expression of COX metabolite receptors.NEW & NOTEWORTHY We demonstrate that the mechanoreflex is sensitized by the cyclooxygenase (COX) pathway within hindlimb skeletal muscles in the rat chronic femoral artery ligation model of simulated peripheral artery disease (PAD). The mechanism of sensitization appears attributable to increased receptors for COX metabolites on sensory neurons and not increased concentration of COX metabolites. Our data may carry important clinical implications for patients with PAD who demonstrate exaggerated increases in blood pressure during exercise compared with healthy counterparts.

PO-126 Inhibition of HIF-1α Alleviates Exaggerated Pressor Response Induced by Static Muscle Contraction in Rats with Peripheral Arterial Disease

Objective Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor mediating adaptive responses to hypoxia and ischemia. A prior work showed that HIF-1α is increased in sensory nerves of rats with femoral artery occlusion. The present study was to examine if the reflex response of blood pressure induced by muscle contraction was altered after injection of HIF-1α inhibitor BAY 87-2243 (BAY87) into the arterial blood supply of the ischemic hindlimb muscles. &#x0D; Methods A rat model of femoral artery ligation was used to study peripheral artery disease. Western blot analysis was employed to examine the protein levels of HIF-1α in the dorsal root ganglion (DRG) tissues. The exercise pressor reflex was evoked by static muscle contraction. &#x0D; Results HIF-1α was increased in the DRG of occluded limbs (optical density: 0.89±0.13 in control vs. 1.5±0.05 in occlusion; P &lt; 0.05, n=6 in each group). Arterial injection of BAY87 (0.2 mg/kg) inhibited expression of HIF-1α in the DRG of occluded limbs three hours following its injection (optical density: 1.02±0.09 in occluded limbs with BAY87 vs. 1.06±0.1 in control limbs; P &gt; 0.05, n=5 in each group). In addition, muscle contraction evoked a greater increase in blood pressure in occluded rats. BAY87 attenuated the enhanced pressor response in occluded rats to a greater degree than in control rats.&#x0D; Conclusions Inhibition of HIF-1α alleviates exaggeration of the exercise pressor reflex in rats under ischemic circumstances of the hindlimbs induced by femoral artery occlusion. &#x0D;

Aryl Hydrocarbon Receptor Nuclear Translocator in Vascular Smooth Muscle Cells Is Required for Optimal Peripheral Perfusion Recovery.

BackgroundLimb ischemia resulting from peripheral vascular disease is a common cause of morbidity. Vessel occlusion limits blood flow, creating a hypoxic environment that damages distal tissue, requiring therapeutic revascularization. Hypoxia‐inducible factors (HIFs) are key transcriptional regulators of hypoxic vascular responses, including angiogenesis and arteriogenesis. Despite vascular smooth muscle cells’ (VSMCs’) importance in vessel integrity, little is known about their functional responses to hypoxia in peripheral vascular disease. This study investigated the role of VSMC HIF in mediating peripheral ischemic responses.Methods and ResultsWe used Arnt SMKO mice with smooth muscle–specific deletion of aryl hydrocarbon receptor nuclear translocator (ARNT, HIF‐1β), required for HIF transcriptional activity, in a femoral artery ligation model of peripheral vascular disease. Arnt SMKO mice exhibit impaired perfusion recovery despite normal collateral vessel dilation and angiogenic capillary responses. Decreased blood flow manifests in extensive tissue damage and hypoxia in ligated limbs of Arnt SMKO mice. Furthermore, loss of aryl hydrocarbon receptor nuclear translocator changes the proliferation, migration, and transcriptional profile of cultured VSMCs. Arnt SMKO mice display disrupted VSMC morphologic features and wrapping around arterioles and increased vascular permeability linked to decreased local blood flow.ConclusionsOur data demonstrate that traditional vascular remodeling responses are insufficient to provide robust peripheral tissue reperfusion in Arnt SMKO mice. In all, this study highlights HIF responses to hypoxia in arteriole VSMCs critical for the phenotypic and functional stability of vessels that aid in the recovery of blood flow in ischemic peripheral tissues.

A modified rat model of hindlimb ischemia for augmentation and functional measurement of arteriogenesis.

Arteriogenesis (collateral artery development) is an adaptive pathway critical for salvage of tissue in the setting of arterial occlusion. Rodent models of arteriogenesis typically involve an experimental occlusion (ligation) of a hindlimb artery and then rely on indirect measures such as laser Doppler perfusion imaging to assess blood flow recovery. Unfortunately, the more commonly utilized measures of distal tissue perfusion at rest are unable to account for hemodynamic and vasoactive variables and thus provide an incomplete assessment of collateral network capacity. We provide a detailed description of modifications to the commonly used model of femoral artery ligation. These serve to alter and then directly assess collateral network’s hemodynamic capacity. By incorporating an arteriovenous fistula distal to the arterial ligation, arterial growth is maximized. Hindlimb perfusion may be isolated to measure minimum resistance of flow around the arterial occlusion, which provides a direct measure of collateral network capacity. Our results reinforce that arteriogenesis is driven by hemodynamic variables, and it can be reliably augmented and measured in absolute terms. Using these modifications to a widely used model, functional arteriogenesis may be more directly studied.

Procalcitonin Impairs Endothelial Cell Function and Viability.

Procalcitonin is used as a diagnostic tool for the identification and risk stratification of septic patients. Procalcitonin plasma concentrations tightly correlate with the severity of the ongoing inflammatory reaction and can rise up to 10,000-fold. Impairment of endothelial cell function plays an important role in the pathogenesis of hypotension and disturbed organ perfusion during sepsis. We investigated the possible effects of procalcitonin itself on endothelial cell function and viability. Human endothelial cells were exposed to 0.01 to 100 ng/mL procalcitonin and investigated for endothelial permeability using transwells, migration in a scratch wound assay and new capillary formation on extracellular matrix in vitro. Tumor necrosis factor-α and vascular endothelial growth factor served as positive controls. Procalcitonin's impact on the response of endothelial cells toward ischemia was investigated in vivo in the murine model of unilateral femoral artery ligation. Procalcitonin-exposed endothelial cells were subjected to immunoblot for the investigation of vascular endothelial-cadherin expression and angiogenic signaling pathways. Flow cytometry was used for the detection of inflammatory activation and viability, and genomic analysis was performed. Data are presented as difference in means and 95% confidence intervals; statistical analyses were performed using analysis of variance/Bonferroni, and P values are reported as adjusted for multiple comparisons (Padjust). Tumor necrosis factor-α and 0.1 ng/mL procalcitonin induced endothelial barrier disruption after incubation of endothelial monolayers for 6 hours (-2.53 [-4.16 to -0.89], P = .0008 and -2.09 [-3.73 to -0.45], Padjust = .0064 compared with vehicle-treated control, respectively). Procalcitonin beginning at concentrations of 0.02 ng/mL reduced endothelial cell migration (0.26 [0.06 to 0.47], Padjust = .0069) and new capillary formation in vitro (0.47 [0.28 to 0.66], Padjust < .0001) contrasting the proangiogenic action of vascular endothelial growth factor. Left ventricular injection of procalcitonin in mice on postoperative day 1, 3, and 5 after induction of ischemia impaired new capillary formation and recovery of hindlimb perfusion in vivo (number of capillaries/mm in the ischemic leg of vehicle-treated versus procalcitonin-treated mice, 852.6 [383.4-1322], Padjust = .0002). Twenty-four-hour incubation with procalcitonin reduced the expression of vascular endothelial-cadherin at 100 ng/mL (0.39 [0.06-0.71], Padjust = .0167) and induced endothelial cell death (apoptosis, -5.4 [-10.67 to -0.13], Padjust = .0431). No alteration in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or extracellular signal-regulated kinase 1/2, and AKT signaling pathways was observed. Genomic analysis revealed regulation of a variety of genes involved in inflammation, angiogenesis, and cell growth. This study found that procalcitonin itself impaired several aspects of endothelial cell function. Procalcitonin-induced loss of endothelial barrier function may contribute to capillary leakage and therapy-refractory hypotension during sepsis. Anti-angiogenic properties of procalcitonin at low concentrations could also identify procalcitonin as a mediator of vascular disease associated with the metabolic syndrome. Future studies are needed to further test procalcitonin as a potential therapeutic target for preserving vascular dysfunction during acute and chronic inflammatory disorders.

Proteinase-Activated Receptor-2 Sensitivity of Amplified TRPA1 Activity in Skeletal Muscle Afferent Nerves and Exercise Pressor Reflex in Rats with Femoral Artery Occlusion

Background/Aims: Limb ischemia occurs in peripheral artery disease (PAD). Sympathetic nerve activity (SNA) that regulates blood flow directed to the ischemic limb is exaggerated during exercise in this disease, and transient receptor potential channel A1 (TRPA1) in thin-fiber muscle afferents contributes to the amplified sympathetic response. The purpose of the present study was to determine the role of proteinase-activated receptor-2 (PAR2) in regulating abnormal TRPA1 function and the TRPA1-mediated sympathetic component of the exercise pressor reflex. Methods: A rat model of femoral artery ligation was employed to study PAD. Dorsal root ganglion (DRG) tissues were obtained to examine the protein levels of PAR2 using western blot analysis. Current responses induced by activation of TRPA1 in skeletal muscle DRG neurons were characterized using whole-cell patch clamp methods. The blood pressure response to static exercise (i.e., muscle contraction) and stimulation of TRPA1 was also examined after a blockade of PAR2. Results: The expression of PAR2 was amplified in DRG neurons of the occluded limb, and PAR2 activation with SL-NH2 (a PAR2 agonist) increased the amplitude of TRPA1 currents to a greater degree in DRG neurons of the occluded limb. Moreover, FSLLRY-NH2 (a PAR antagonist) injected into the arterial blood supply of the hindlimb muscles significantly attenuated the pressor response to muscle contraction and TRPA1 stimulation in rats with occluded limbs. Conclusions: The PAR2 signal in muscle sensory nerves contributes to the amplified exercise pressor reflex via TRPA1 mechanisms in rats with femoral artery ligation. These findings provide a pathophysiological basis for autonomic responses during exercise activity in PAD, which may potentially aid in the development of therapeutic approaches for improvement of blood flow in this disease.

Suppression of ischemia in arterial occlusive disease by JNK-promoted native collateral artery development.

Arterial occlusive diseases are major causes of morbidity and mortality. Blood flow to the affected tissue must be restored quickly if viability and function are to be preserved. We report that disruption of the mixed-lineage protein kinase (MLK) - cJun NH2-terminal kinase (JNK) signaling pathway in endothelial cells causes severe blockade of blood flow and failure to recover in the murine femoral artery ligation model of hindlimb ischemia. We show that the MLK-JNK pathway is required for the formation of native collateral arteries that can restore circulation following arterial occlusion. Disruption of the MLK-JNK pathway causes decreased Dll4/Notch signaling, excessive sprouting angiogenesis, and defects in developmental vascular morphogenesis. Our analysis demonstrates that the MLK-JNK signaling pathway is a key regulatory mechanism that protects against ischemia in arterial occlusive disease.

HIF-regulated HO-1 gene transfer improves the post-ischemic limb recovery and diminishes TLR-triggered immune responses — Effects modified by concomitant VEGF overexpression

Heme oxygenase-1 (HO-1) mitigates cellular injury by antioxidant, anti-apoptotic, anti-inflammatory and proangiogenic effects. Vascular endothelial growth factor (VEGF) is a critical regulator of blood vessel growth. Their coordinated action was analyzed in a model of femoral artery ligation (FAL) in mice lacking HO-1 gene (HO-1 KO). Gastrocnemius skeletal muscles of HO-1 KO mice were preemptively injected with plasmids containing hypoxia-response element (HRE) driving the expression of only HO-1 (pHRE-HO1) or both HO-1 and VEGF (pHRE-HO1–VEGF). At day 14th the pHRE-HO1 vector increased an impaired post-ischemic blood flow recovery in HO-1 KO mice to the level observed in wild-type (WT) mice subjected to FAL and pHRE-HO1–VEGF restored it already at day 7. The pHRE-HO1 gene therapy diminished, when compared to control pHRE-empty-treated HO-1 KO mice, the expression of toll-like receptors (TLR4 and TLR9) and inflammatory cytokines (IL-1β, IL-6 and TNFα) at day 3, whereas opposite effects were observed following concomitant HO-1 and VEGF gene transfer. Moreover, HO-1 diminished ischemia-induced expression of MyoD involved in satellite cell differentiation in HO-1 KO mice. Our results confirm the therapeutic potential of HO-1 and VEGF against critical limb ischemia although, their concomitant delivery may have contradictory actions on the resolution of inflammation.

Abstract W MP34: Magnetically-Enhanced Diffusion (MED) of Intravenous tPA for Acute Ischemic Stroke: Preclinical Data

Background: Diffusion of drug to the clot in a no flow environment may be a major factor in the relative ineffectiveness of iv-tPA. The MED system uses an external magnet and magnetic particles (MP) to improve IV-tPA diffusion to clots and to mix tPA at the clot surface. Methods: Diffusion experiments were conducted in a femoral artery ligation model using Evan’s Blue dye as surrogate to tPA. Clot surface interaction experiments were conducted in a jugular vein rabbit model using thrombin-induced occlusions. tPA was locally delivered. Time to recanalization was measured out to 3hrs for 312.5U to 5000U of tPA in ~500uL of blood (0.6U/uL and 9.9U/uL, respectively), with and without MP. Results: Femoral dye studies showed a &gt;5X faster diffusion using the MED system in the femoral artery (Figure 1). Jugular lysis studies showed greater variability in the tPA-alone arm, which possessed several failures to recanalize. No recanalization occurred in &lt;30min for tPA doses &lt;2500U. &lt;25% of the 625U doses restored flow in &lt;1hr. Use of the MED system substantially improved tPA lysis reliability (all recanalization &lt;140min for all doses). 67% of the 625U doses restored flow &lt;1hr. Compared to successful tPA recanalization alone, scatter was significantly smaller and time-to-recanalization 2.5X faster (Figure 2). Conclusion: This study demonstrates the ability of the MED system to accelerate both tPA transport to a clot and clot lysis. This observation supports efficacy signals seen in the human feasibility trial. These results may provide a new understanding of the variability historically seen in thrombolytic efficacy, which may be related to inefficient thrombolytic contact with the thrombus.

Abstract 11611: Muscle Superoxide Dismutase and Augmented Autonomic Responsiveness in Rats With Peripheral Artery Disease

Background: During exercise, responses of sympathetic nerve and blood pressure are amplified in patients with peripheral artery disease (PAD). This worsens the restricted blood flow directed to the limbs in this disease. The purpose of this study was to determine the role played by muscle oxidative stress in regulating the augmented autonomic responses to static exercise in PAD. Superoxide dismutases (SOD) is a class of enzymes to catalyze the dismutation of superoxide into oxygen and hydrogen peroxide, and considered as an important antioxidant. Thus, our specific hypothesis was that limb ischemia impairs muscle SOD and thereby leads to abnormal autonomic responsiveness observed in PAD. Methods and Results: We used a rat model of femoral artery ligation to study PAD. Note that all data are presented as mean ± SE. First, western blot analysis shows that 72 hours of femoral occlusion significantly decreased the protein levels of SOD in the oxidative muscle (optical density: 1.02±0.03 in control vs. 0.21±0.02 after ligation, P&lt; 0.05; n = 6 in each group) and glycolytic muscle (optical density: 1.03±0.03 in control vs. 0.65±0.04 after ligation, P&lt; 0.05; n = 6 in each group). Additional immunofluorescence study demonstrates down-regulation of SOD in the ischemic hindlimb muscles. Second, the reflex blood pressure response to static muscle contraction was examined in control rats (n=5) and rats with 72 hours of femoral occlusion (n=6). The data show that the augmented reflex pressor response to muscle contraction was significantly attenuated after compensating SOD in ischemic muscles by chronic administration of tempol (a mimetic of SOD, 30 mg over a period of 72 hours) into the hindlimb. In the ligated rats, mean arterial pressure response was 26±3 mmHg with no tempol (n=6) and 12±2 mmHg with tempol application ( P&lt; 0.05 vs. group with no tempol; n =5). Conclusions: Muscle SOD is blunted in the ischemic limbs and supplement of SOD mimetic for anti-oxidation improves the augmented blood pressure response during static exercise, which is beneficial to the restricted blood flow to the limbs in PAD patients.