PER1 promotes functional recovery of mice with hindlimb ischemia by inducing anti-inflammatory macrophage polarization.

Hindlimb ischemia (HLI) is an arterial occlusive disease that exposes the patients to the risk of limb gangrene and loss. Polarization of macrophages is related to HLI-induced inflammation. Period circadian regulator 1 (PER1) is a core component of the circadian clock. We first showed, based upon bioinformatics analysis of microarray data, that PER1 expression was reduced in monocytes from patients with critical limb ischemia. The proximal femoral artery in the left hindlimb of male mice was ligated and then the femoral artery and its collateral branches were removed to establish the HLI mouse model. After modeling, a single intramuscular injection of 1 × 109 pfu Ad-PER1 was performed at the adductor and gastrocnemius muscles. The gastrocnemius muscle tissues were collected at day 0, 3, 7, 14, 21 post-HLI. There was obvious pathological necrosis, accompanied with reduced expression of PER1 in the muscle tissues of HLI mice. Expression of CD68 and CD31 seemed to be corresponded to PER1 in gastrocnemius muscle, implying the potential of PER1 in regulating macrophage-related inflammation and angiogenesis. PER1 overexpression diminished myocyte damage, promoted blood flow restoration and improved behavioral scores of HLI mice. Immunostaining of CD31 and α-SMA revealed that PER1 upregulation reversed HLI-induced decreases in capillary and arteriole density. In vitro, RAW264.7 cells were cultured in hypoxia (1% O2) for 24 h. The percentage of pro-inflammatory CD86+ macrophages (M1 type) was decreased and that of anti-inflammatory CD206+ macrophages (M2 type) was increased when PER1 was overexpressed. Moreover, the expression levels of TNF-α, IL-6 and M1-type marker iNOS were decreased, and levels of IL-10 and M2-type marker Arg-1 were increased by PER1 in gastrocnemius muscle of HLI mice and hypoxia-treated RAW264.7 cells. PER1 might reduce M1 macrophage polarization and promote M2 macrophage polarization, and thus exert anti-inflammatory and pro-angiogenic actions. Our findings suggest that PER1 overexpression promotes functional recovery of mice with HLI through regulating macrophage polarization.

CRS induces depression-like behavior after MCAO in rats possibly by activating p38 MAPK.

Post-stroke depression (PSD) is a common neuropsychiatric complication of stroke, which seriously affects the quality of life and prognosis of patients. Nevertheless, the pathogenesis of PSD remains unclear. In our study, a PSD rat model was established by chronic restraint stress (CRS) combined with middle cerebral artery occlusion (MCAO). Depressive and anxiety-like behaviors were tested, as well as Neuronal loss and Apoptosis. The expression of synapse and p38 MAPK signaling pathway -relevant proteins was detected. Our data indicated that CRS combined with MCAO could induce depression-like and anxiety-like behaviors, which led to neuronal damage, apoptosis, and cellular loss in the left parietal cortex and left hippocampus. Furthermore, CRS combined with MCAO decreased synaptic plasticity in the parietal cortex and left hippocampus. We found that CRS combined with MCAO had activated the p38 MAPK signaling pathway, and decreased the expression of pathway-related proteins MKK6 and MKK3. These results suggested that CRS combined with MCAO could lead to depression-like behavior via neuronal damage, apoptosis and reduced synaptic plasticity, which might be related to the activation of the p38 MAPK pathway. Therefore, it provides novel ideas for the research on the intervention and prevention mechanisms of PSD.

PHACTR-1 (Phosphatase and Actin Regulator 1) Deficiency in Either Endothelial or Smooth Muscle Cells Does Not Predispose Mice to Nonatherosclerotic Arteriopathies in 3 Transgenic Mice.

BACKGROUND: Genome-wide association studies have revealed robust associations of common genetic polymorphisms in an intron of the PHACTR-1 (phosphatase and actin regulator 1) gene (chr6p24), with cervical artery dissection, spontaneous coronary artery dissection, and fibromuscular dysplasia. The aim was to assess its role in the pathogenesis of cervical artery dissection or fibromuscular dysplasia. METHODS: Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells using 2 tissue-specific Cre-driver (PDGFB [platelet-derived growth factor B]-CreERT2 mice and Tie2 [tyrosine kinase with immunoglobulin and EGF homology domains]-Cre) and smooth muscle cells (smooth muscle actin-CreERT2) with a third tissue-specific Cre-driver. RESULTS: To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed first, a decrease in Phactr1 transcription and Phactr1 expression in endothelial cell and smooth muscle cell isolated from Phactr1iPDGFB and Phactr1iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology, or actin organization were not different in knockout mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin II-induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there were no more consequences of Phactr1 deletion during embryogenesis in endothelial cells. CONCLUSIONS: Loss of PHACTR-1 function in the cells involved in vascular physiology does not appear to induce a pathological vascular phenotype. The in vivo effect of the intronic variation described in genome-wide association studies is unlikely to involve downregulation in PHACTR-1 expression.

KANK4 Promotes Arteriogenesis by Potentiating VEGFR2 Signaling in a TALIN-1-Dependent Manner.

BACKGROUND: Arteriogenesis plays a critical role in maintaining adequate tissue blood supply and is related to a favorable prognosis in arterial occlusive diseases. Strategies aimed at promoting arteriogenesis have thus far not been successful because the factors involved in arteriogenesis remain incompletely understood. Previous studies suggest that evolutionarily conserved KANK4 (KN motif and ankyrin repeat domain-containing proteins 4) might involve in vertebrate vessel development. However, how the KANK4 regulates vessel function remains unknown. We aim to determine the role of endothelial cell-specifically expressed KANK4 in arteriogenesis. METHODS: The role of KANK4 in regulating arteriogenesis was evaluated using Kank4-/- and KANK4iECOE mice. Molecular mechanisms underlying KANK4-potentiated arteriogenesis were investigated by employing RNA transcriptomic profiling and mass spectrometry analysis. RESULTS: By analyzing Kank4-EGFP reporter mice, we showed that KANK4 was specifically expressed in endothelial cells. In particular, KANK4 displayed a dynamic expression pattern from being ubiquitously expressed in all endothelial cells of the developing vasculature to being explicitly expressed in the endothelial cells of arterioles and arteries in matured vessels. In vitro microfluidic chip-based vascular morphology analysis and in vivo hindlimb ischemia assays using Kank4-/- and KANK4iECOE mice demonstrated that deletion of KANK4 impaired collateral artery growth and the recovery of blood perfusion, whereas KANK4 overexpression leads to increased vessel caliber and blood perfusion. Bulk RNA sequencing and Co-immunoprecipitation/mass spectrometry (Co-IP/MS) analysis identified that KANK4 promoted EC proliferation and collateral artery remodeling through coupling VEGFR2 (vascular endothelial growth factor receptor 2) to TALIN-1, which augmented the activation of the VEGFR2 signaling cascade. CONCLUSIONS: This study reveals a novel role for KANK4 in arteriogenesis in response to ischemia. KANK4 links VEGFR2 to TALIN-1, resulting in enhanced VEGFR2 activation and increased EC proliferation, highlighting that KANK4 is a potential therapeutic target for promoting arteriogenesis for arterial occlusive diseases.

Arteriogenesis and Therapeutic Angiogenesis-An Update.

Vascular occlusive diseases such myocardial infarction, peripheral artery disease of the lower extremities, or stroke still represent a substantial health burden worldwide [...].

Role of Vascular Smooth Muscle Cell Phenotype Switching in Arteriogenesis.

Arteriogenesis is one of the primary physiological means by which the circulatory collateral system restores blood flow after significant arterial occlusion in peripheral arterial disease patients. Vascular smooth muscle cells (VSMCs) are the predominant cell type in collateral arteries and respond to altered blood flow and inflammatory conditions after an arterial occlusion by switching their phenotype between quiescent contractile and proliferative synthetic states. Maintaining the contractile state of VSMC is required for collateral vascular function to regulate blood vessel tone and blood flow during arteriogenesis, whereas synthetic SMCs are crucial in the growth and remodeling of the collateral media layer to establish more stable conduit arteries. Timely VSMC phenotype switching requires a set of coordinated actions of molecular and cellular mediators to result in an expansive remodeling of collaterals that restores the blood flow effectively into downstream ischemic tissues. This review overviews the role of VSMC phenotypic switching in the physiological arteriogenesis process and how the VSMC phenotype is affected by the primary triggers of arteriogenesis such as blood flow hemodynamic forces and inflammation. Better understanding the role of VSMC phenotype switching during arteriogenesis can identify novel therapeutic strategies to enhance revascularization in peripheral arterial disease.

The slope of cerebral oxyhemoglobin oscillation is associated with vascular reserve capacity in large artery steno-occlusion.

Inadequate cerebral perfusion is a risk factor for cerebral ischemia in patients with large artery steno-occlusion. We investigated whether prefrontal oxyhemoglobin oscillation (ΔHbO2, 0.6-2 Hz) was associated with decreased vascular reserve in patients with steno-occlusion in the large anterior circulation arteries. Thirty-six patients with steno-occlusion in the anterior circulation arteries (anterior cerebral artery, middle cerebral artery, and internal carotid artery) were included and compared to thirty-six control subjects. Patients were categorized into two groups (deteriorated vascular reserve vs. preserved vascular reserve) based on the results of Diamox single- photon emission computed tomography imaging. HbO2 data were collected using functional near-infrared spectroscopy. The slope of ΔHbO2 and the ipsilateral/contralateral slope ratio of ΔHbO2 were analyzed. Among the included patients (n = 36), 25 (69.4%) had deteriorated vascular reserve. Patients with deteriorated vascular reserve had a significantly higher average slope of ΔHbO2 on the ipsilateral side (5.01 ± 2.14) and a higher ipsilateral/contralateral ratio (1.44 ± 0.62) compared to those with preserved vascular reserve (3.17 ± 1.36, P = 0.014; 0.93 ± 0.33, P = 0.016, respectively) or the controls (3.82 ± 1.69, P = 0.019; 0.94 ± 0.29, P = 0.001). The ipsilateral/contralateral ΔHbO2 ratio could be used as a surrogate for vascular reserve in patients with severe steno-occlusion in the anterior circulation arteries.

Involvement of Supraoptic Astrocytes in Basilar Artery Occlusion-Evoked Differential Activation of Vasopressin Neurons and Vasopressin Secretion in Rats.

Vasopressin (VP) is a key factor in the development of brain injury in ischemic stroke. However, the regulation of VP secretion in basilar artery occlusion (BAO) remains unclear. To clarify the regulation of VP secretion in BAO and the underlying mechanisms, we performed this study in a rat model of BAO with (BC) or without common carotid artery occlusion (CCAO). The results showed that BAO and BC time-dependently increased neurological scores and that BC also increased water contents in the medulla at 2 h and in the pontine at 8 h. Moreover, plasma VP level increased significantly at BAO-8 h, CCAO and BC-2 h but not at BC-8 h; however, VP expressions increased in the supraoptic nucleus (SON) at BC-8 h. The neurological scores were highly correlated with pontine water contents and plasma VP levels. The number of phosphorylated extracellular signal-regulated protein kinase1/2-positive VP neurons increased significantly in the SON at BC-8 h. Similarly, the number of c-Fos-positive VP neurons increased significantly in the SON at BAO-8 h and BC-8 h. In addition, the length of glial fibrillary acidic protein (GFAP) filaments increased significantly in BC compared to BAO only. Aquaporin 4 (AQP4) puncta around VP neurons increased significantly at BC-8 h relative to BC-2 h, which had negative correlation with plasma VP levels. These findings indicate that BAO facilitates VP secretion and increases VP neuronal activity in the SON. The peripheral VP release is possibly under a negative feedback regulation of central VP neuronal activity through increasing GFAP and AQP4 expression in astrocytic processes.

Towards the Therapeutic Use of Thrombospondin 1/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent.

OBJECTIVE: TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, such as, platelet adhesion to vascular endothelium, nitric oxide/cGMP (cyclic guanosine monophosphate) signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and Results: A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, while Thbs1-/- mice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters. CONCLUSIONS: Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.

Everolimus Rescues the Phenotype of Elastin Insufficiency in Patient Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

OBJECTIVE: Elastin gene deletion or mutation leads to arterial stenoses due to vascular smooth muscle cell (SMC) proliferation. Human induced pluripotent stem cells-derived SMCs can model the elastin insufficiency phenotype in vitro but show only partial rescue with rapamycin. Our objective was to identify drug candidates with superior efficacy in rescuing the SMC phenotype in elastin insufficiency patients. Approach and Results: SMCs generated from induced pluripotent stem cells from 5 elastin insufficiency patients with severe recurrent vascular stenoses (3 Williams syndrome and 2 elastin mutations) were phenotypically immature, hyperproliferative, poorly responsive to endothelin, and exerted reduced tension in 3-dimensional smooth muscle biowires. Elastin mRNA and protein were reduced in SMCs from patients compared to healthy control SMCs. Fourteen drug candidates were tested on patient SMCs. Of the mammalian target of rapamycin inhibitors studied, everolimus restored differentiation, rescued proliferation, and improved endothelin-induced calcium flux in all patient SMCs except one Williams syndrome. Of the calcium channel blockers, verapamil increased SMC differentiation and reduced proliferation in Williams syndrome patient cells but not in elastin mutation patients and had no effect on endothelin response. Combination treatment with everolimus and verapamil was not superior to everolimus alone. Other drug candidates had limited efficacy. CONCLUSIONS: Everolimus caused the most consistent improvement in SMC differentiation, proliferation and in SMC function in patients with both syndromic and nonsyndromic elastin insufficiency, and offers the best candidate for drug repurposing for treatment of elastin insufficiency associated vasculopathy.

The Association of Periodontitis and Peripheral Arterial Occlusive Disease-A Systematic Review.

Background: Observational studies support an association between periodontitis (PD) and atherosclerotic vascular disease, but little is known specifically about peripheral arterial occlusive disease (PAOD). OBJECTIVES: To systematically review the evidence for an association between PD and PAOD. DATA SOURCES: Medline via PubMed. REVIEW METHODS: We searched the Pubmed database for original studies, case reports, case series, meta-analyses and systematic reviews that assessed whether there is an association between PD (all degrees of severity) and PAOD (all degrees of severity). The reporting of this systematic review was in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement following the Population, Intervention, Control, and Outcome (PICO) format. RESULTS: 17 out of 755 detected studies were included in the qualitative synthesis. Nine studies demonstrated associations between PD and PAOD, and two studies reported associations between tooth loss and PAOD. Six studies addressed the pathomechanism regarding PD as a possible trigger for PAOD. No study that dismissed an association could be detected. Odds ratios or hazard ratios ranged from 1.3 to 3.9 in four large cohort studies after adjusting for established cardiovascular risk factors. CONCLUSIONS: The presented evidence supports a link between PD and PAOD. Further studies which address the temporality of PD and PAOD and randomized controlled intervention trials examining the causal impact of PD on PAOD are needed. Although our results cannot confirm a causal role of PD in the development of PAOD, it is likely that PD is associated with PAOD and plays a contributing role.

[Objective assessment of the degree of ischaemia and efficacy of treatment in chronic arterial insufficiency of lower limbs]. / Ob''ektivnaia otsenka stepeni ishemii i éffektivnosti lecheniia pri khronicheskoi arterial'noi nedostatochnosti nizhnikh konechnostei.

Presented in the review is the data concerning contemporary criteria for assessing the degree of chronic lower limb ischaemia and quality of the treatment performed. Problems regarding objectivization of the degree of an ischaemic lesion of an extremity and assessment of quality of conservative treatment still remain unresolved. Currently, in the world practice along with instrumental methods (assessment of the ankle-brachial index) subjective criteria are mainly employed: assessment of pain-free walking distance or maximum distance walked. In order to work out appropriate regiments of conservative treatment and compare efficacy of various drugs for treatment of chronic lower limb ischaemia and, in particular, intermittent claudication, it is necessary to use objective criteria. Detailed consideration is hence given to the possibilities of using morphological, biochemical and histological criteria such as determination of vascular endothelial growth factor A (VEGF-A) in peripheral blood and determination of apoptosis markers (BNIP3) and hypoxia-inducible factor (HIF-1) in a biopsy sample of the gastrocnemius muscle of the ischaemized extremity.

The Role of Adipokines in Surgical Procedures Requiring Both Liver Regeneration and Vascular Occlusion.

Liver regeneration is a perfectly calibrated mechanism crucial to increase mass recovery of small size grafts from living donor liver transplantation, as well as in other surgical procedures including hepatic resections and liver transplantation from cadaveric donors. Regeneration involves multiple events and pathways in which several adipokines contribute to their orchestration and drive hepatocytes to proliferate. In addition, ischemia-reperfusion injury is a critical factor in hepatic resection and liver transplantation associated with liver failure or graft dysfunction post-surgery. This review aims to summarize the existing knowledge in the role of adipokines in surgical procedures requiring both liver regeneration and vascular occlusion, which increases ischemia-reperfusion injury and regenerative failure. We expose and discuss results in small-for-size liver transplantation and hepatic resections from animal studies focused on the modulation of the main adipokines associated with liver diseases and/or regeneration published in the last five years and analyze future perspectives and their applicability as potential targets to decrease ischemia-reperfusion injury and improve regeneration highlighting marginal states such as steatosis. In our view, adipokines means a promising approach to translate to the bedside to improve the recovery of patients subjected to partial hepatectomy and to increase the availability of organs for transplantation.

Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion.

INTRODUCTION: Lung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. MATERIALS AND METHODS: A total of 24 pigs were randomized in three groups. Group I (n = 8) underwent sham operation, group II (n = 8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n = 8) received 3 doses of lazaroid (3 mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7 t h postoperative day and lung specimens were harvested for molecular analysis. RESULTS: mRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (-63.7%), LTC4S (-35.9%) and iNOS (-60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). CONCLUSION: Lazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.

Hemodynamics and oxygen extraction in chronic large artery steno-occlusive disease: Clinical applications for predicting stroke risk.

Depending on the adequacy of collateral sources of blood flow, arterial stenosis or occlusion may lead to reduced perfusion pressure and ultimately reduced blood flow in the distal territory supplied by that vessel. There are two well-defined compensatory mechanisms to reduced pressure or flow - autoregulatory vasodilation and increased oxygen extraction fraction. Other changes, such as metabolic downregulation, are likely. The positive identification of autoregulatory vasodilation and increased oxygen extraction fraction in humans is an established risk factor for future ischemic stroke in some disease states such as atherosclerotic carotid stenosis and occlusion. The mechanisms by which ischemic stroke may occur are not clear, and may include an increased vulnerability to embolic events. The use of hemodynamic assessment to identify patients with occlusive vasculopathy at an increased risk for stroke is very appealing for several different patient populations, such as those with symptomatic intracranial atherosclerotic disease, moyamoya phenomenon, complete internal carotid artery occlusion, and asymptomatic cervical carotid artery stenosis. While there is very good data for stroke risk prediction in some of these groups, no intervention based on these tools has been proven effective yet. In this manuscript, we will review these topics above and identify areas for future research.

Numerical simulation of low-density lipoprotein mass transport in human arterial stenosis - Calculation of the filtration velocity.

Accumulation of cholesterol and other atherogenic lipids such as low-density lipoprotein (LDL) in artery wall causes reduction of vessel diameter and artery stenosis. The study of the mass transfer of these large molecules in the wall with considering effective factors on lumen flow and different physiological factors is the subject considered nowadays. In this paper, results of two dimensional and axi-symmetric simulations of three different models of the artery with 60% stenosis under pulsatile blood flow are presented. Filtration velocity of LDL mass transport in the permeable artery wall and shear stress of blood flow are investigated using ADINA software Three different flow models are considered. In the first and second models, the filtration velocity considered as a given parameter and constant in arterial wall boundary, while in third model arterial wall considered as porous wall, the filtration velocity is calculated from pressure difference as an input parameter of the model. The results show that filtration velocity is strongly depend on geometry and it is not constant along the wall, contrary to simplified models. The results of concentration variations in lumen and wall illustrate the increase in near wall LDL concentration or concentration polarization.

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.

TRPA1 Function in Skeletal Muscle Sensory Neurons Following Femoral Artery Occlusion.

BACKGROUND/AIMS: Transient receptor potential channel A1 (TRPA1) is engaged in amplified autonomic responses evoked by stimulation of muscle afferent nerves in rats with experimental peripheral arterial disease. The purposes of this study were to characterize current responses induced by activation of TRPA1 in dorsal root ganglion (DRG) neurons of control limbs and limbs with femoral artery occlusion. METHODS: DRG neurons from rats were labeled by injecting the fluorescence tracer DiI into the hindlimb muscles and whole-cell patch clamp experiments were performed to determine TRPA1 currents. RESULTS: Data show that AITC (a TRPA1 agonist) from the concentrations of 50 µM to 200 µM produces a dose-dependent increase of amplitudes of inward current responses. Notably, the peak current amplitude induced by AITC is significantly larger in DRG neurons of ligated limbs than that in control limbs. AITC-induced current responses are observed in small and medium size DRG neurons, and there is no difference in size distribution of DRG neurons between control limbs and ligated limbs. However, femoral occlusion increases the percentage of the AITC-sensitive DRG neurons as compared to control. AITC-induced currents in DRG neurons are significantly attenuated by exposure to 10 µM of HC-030031, a potent and selective inhibitor of TRPA1, in both control and femoral occlusion groups. In addition, capsaicin (a TRPV1 agonist) evokes a greater increase in the amplitude of AITC-currents in DRG neurons of ligated limbs than that in control limbs. CONCLUSIONS: A greater current response with activation of TRPA1 is developed in muscle afferent nerves when hindlimb arterial blood supply is deficient under ischemic conditions; and TRPV1 is partly responsible for augmented TRPA1 responses induced by arterial occlusion.