Flow-induced Dilation In Arterioles Research Articles

Abstract 15676: Sex Differences in Leg Microvascular Arteriolar Function in Patients With Limb Ischemia

Introduction: There are known sex differences in peripheral artery disease (PAD) symptom presentation with women manifesting atypical symptoms and a higher vascular burden. Women are also more prone to periprocedural complications possibly due to sex-specific endothelial dysfunction and microvascular impairment. There is a critical need to understand sex specific differences in microvascular function to improve outcomes for women with PAD. Hypothesis: Lower limb arteriolar flow induced dilation (FID) will be significantly reduced in women, compared to men. Methods: Combined muscle and fat tissue were obtained from the affected leg in patients undergoing revascularization due to PAD. Arterioles were dissected, cannulated, and suspended in a physiological chamber at a stable intraluminal pressure. FID was assessed at increasing intraluminal pressure gradients. A subsample of vessels underwent arteriolar dilation with ACh with and without L-NAME, and SNP. Results: We quantified arteriolar FID in 24 arterioles (7=not viable) from 18 patients undergoing revascularization due to PAD (age 60.2 + 7.4, 44%=F, 61%=Black). Heterogeneity was observed in FID with significantly reduced response observed in women. FID in women at 60 cmH 2 O was 24.7 + 20.5%, compared to 52.6 + 28.0% in men, p=.05, and AUC was 69.0 + 66.8 in women, compared to 147.6 + 82.0 in men (p=.07). Findings were similar in both muscle and adipose arterioles. Vessels treated with Ach responded similar to FID. Vessels treated with L-NAME and SNP showed variable blunted response. Conclusions: Women had more impairment in arteriolar function compared to men, providing insight into sex dimorphism in the PAD population. Findings indicated that FID in arterioles is mostly nitric oxide dependent, although patient and sex specific changes in vascular smooth muscle signaling/function are present. This study support the need to develop microvascular and sex specific interventions for patients with PAD.

Investigation of the Dual Functional Role of Ceramide in the Human Microcirculation

Increased plasma ceramide levels and microvascular dysfunction are both independent risk factors for major adverse cardiac events (MACE). We have previously shown that chronic exposure to exogenous ceramide promotes microvascular endothelial dysfunction, defined as hydrogen peroxide (H2O2)-mediated flow-induced dilation (FID) as opposed to dilation due to formation and release of endothelial nitric oxide (NO). Interestingly, ceramide and its metabolites (e.g. sphingosine-1-phosphate; S1P) have also been shown to stimulate production of NO. Our previous studies indicate that activation of the ceramide-producing enzyme neutral sphingomyelinase (NSmase) in necessary for NO-mediated FID. We therefore hypothesized that formation of S1P is responsible for cellular increases in NO from both acute exogenous administration as well as endogenous formation of ceramide from shear. Human resistance arterioles (100-200µm) were dissected from adipose tissue collected from healthy patients and were prepared for videomicroscopy. Increasing doses of exogenous C2 ceramide (10-9 to 10-5 M) were administered in the absence or presence of the NO scavenger c-PTIO (1µM) and a sphingosine kinase inhibitor (SpKi, 5µM). We observed a dose-response increase in arteriolar dilation from ceramide (46.9%±10.3 of maximal dilator capacity±SEM, n=3) that was impaired by c-PTIO (15%±6.0, n=3). Ceramide-induced dilation was also decreased in the presence of the SpKi (20.8%±4.0, n=3). To examine the role of S1P formation in NO-mediated FID, SpKi was administered to healthy arterioles (5µM, 30 min) prior to initiating flow (pressure gradient 5-100cm H2O) and a dramatic decrease in overall dilation was observed (7.7%±8.0, n=3). Together these findings suggest that NO generated from ceramide is primarily due to the formation of S1P, a process also critical to maintain FID in arterioles from healthy individuals.

Is a Biomarker Microvascular Model for Dilation?

Does low-fat diet for weight loss make the impaired microvascular dilation improved through switching to nitric oxide (NO)-dependent in obese adults? The answer is yes. Shane Phillips research group has tested this hypothesis through their most recent publication [1]. While systemic NO doesn’t change, localized NO was increased after a six-week low-fat diet for weight loss (~25% calorie deficit) but not for weight maintenance to obsess subjects with body mass index (BMI most subjects in 30-35, and only a few in 35-40 kg/m2) through increasing flow-induced dilation of arterioles. These in vitro microvascular model data are consistent with data with clinical increase of flow-mediated dilation and decrease of BMI in post- compared to pre-low-fat diet for weight loss

Arginase Promotes Skeletal Muscle Arteriolar Endothelial Dysfunction in Diabetic Rats

Endothelial dysfunction is a characteristic feature in diabetes that contributes to the development of vascular disease. Recently, arginase has been implicated in triggering endothelial dysfunction in diabetic patients and animals by competing with endothelial nitric oxide synthase for substrate l-arginine. While most studies have focused on the coronary circulation and large conduit blood vessels, the role of arginase in mediating diabetic endothelial dysfunction in other vascular beds has not been fully investigated. In the present study, we determined whether arginase contributes to endothelial dysfunction in skeletal muscle arterioles of diabetic rats. Diabetes was induced in male Sprague Dawley rats by streptozotocin injection. Four weeks after streptozotocin administration, blood glucose, glycated hemoglobin, and vascular arginase activity were significantly increased. In addition, a significant increase in arginase I and II mRNA expression was detected in gracilis muscle arterioles of diabetic rats compared to age-matched, vehicle control animals. To examine endothelial function, first-order gracilis muscle arterioles were isolated, cannulated in a pressure myograph system, exposed to graded levels of luminal flow, and internal vessel diameter measured. Increases in luminal flow (0–50 μL/min) caused progressive vasodilation in arterioles isolated from control, normoglycemic animals. However, flow-induced vasodilation was absent in arterioles obtained from streptozotocin-treated rats. Acute in vitro pretreatment of blood vessels with the arginase inhibitors Nω-hydroxy-nor-l-arginine or S-(2-boronoethyl)-l-cysteine restored flow-induced responses in arterioles from diabetic rats and abolished differences between diabetic and control animals. Similarly, acute in vitro pretreatment with l-arginine returned flow-mediated vasodilation in vessels from diabetic animals to that of control rats. In contrast, d-arginine failed to restore flow-induced dilation in arterioles isolated from diabetic animals. Administration of sodium nitroprusside resulted in a similar degree of dilation in arterioles isolated from control or diabetic rats. In conclusion, the present study identifies arginase as an essential mediator of skeletal muscle arteriolar endothelial dysfunction in diabetes. The ability of arginase to induce endothelial dysfunction in skeletal muscle arterioles may further compromise glucose utilization and facilitate the development of hypertension in diabetes.

NADPH oxidase‐dependent reactive oxygen species are involved in flow‐induced dilation of human adipose arterioles

Hydrogen peroxide (H2O2) as an endothelium‐derived vasodilator factor mediates flow‐induced dilation (FID) in human adipose arterioles from patients with coronary artery disease (CAD). The intracellular sources responsible for H2O2 generation during FID remain unclear. This study is designed to test whether NADPH oxidase (NOX) serves as a potential source of reactive oxygen species (ROS) formation contributing to FID. Visceral adipose arterioles (~150 μm ID) obtained from patients with or without CAD were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. After constriction with endothelin‐1, arterioles were subjected to stepwise increase in luminal flow before and after treatment with gp91‐ ds‐tat, a cell‐permeable peptide inhibitor of NOX, or a scrambled control peptide. Flow induced a graded dilation of adipose arterioles from CAD (maximal dilation: 61±3%, n=6), and this dilation was markedly reduced by gp91‐ds‐tat (50μM; 5±4%, n=5, P<0.05). FID was not affected by the scrambled peptide (67±2 vs. 61±3% of controls). In contrast to arterioles from CAD patients, gp91‐ds‐tat had no effect on FID in arterioles from patients without CAD (68±2% vs. 67±2% of controls, n=5–6). In conclusion, NOX plays an essential role in FID of human adipose arterioles.

Estrogen replacement restores flow-induced vasodilation in coronary arterioles of aged and ovariectomized rats

The risk for cardiovascular disease (CVD) increases with advancing age; however, the age at which CVD risk increases significantly is delayed by more than a decade in women compared with men. This cardioprotection, which women experience until menopause, is presumably due to the presence of ovarian hormones, in particular, estrogen. The purpose of this study was to determine how age and ovarian hormones affect flow-induced vasodilation in the coronary resistance vasculature. Coronary arterioles were isolated from young (6 mo), middle-aged (14 mo), and old (24 mo) intact, ovariectomized (OVX), and ovariectomized + estrogen replaced (OVE) female Fischer-344 rats to assess flow-induced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 +/- 4 vs. old: 34 +/- 6; % relaxation). Ovariectomy reduced flow-induced dilation in arterioles from young females, and estrogen replacement restored vasodilation to flow. In aged females, flow-induced vasodilation of arterioles was unaltered by OVX; however, estrogen replacement improved flow-induced dilation by approximately 160%. The contribution of nitric oxide (NO) to flow-induced dilation, assessed by nitric oxide synthase (NOS) inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME), declined with age. l-NAME did not alter flow-induced vasodilation in arterioles from OVX rats, regardless of age. In contrast, l-NAME reduced flow-induced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flow-induced, NO-mediated dilation in coronary arterioles of female rats is related, in part, to a loss of ovarian estrogen, and estrogen supplementation can improve flow-induced dilation, even at an advanced age.

COX-2 contributes to the maintenance of flow-induced dilation in arterioles of eNOS-knockout mice

Our previous studies demonstrated that, in gracilis muscle arterioles of male mice deficient in the gene for endothelial nitric oxide synthase (eNOS), flow-induced dilation (FID) is mediated by endothelial PGs. Thus the present study aimed to identify the specific isoform of cyclooxygenase (COX) responsible for the compensatory mediation of FID in arterioles of eNOS-knockout (KO) mice. Experiments were conducted on gracilis muscle arterioles of male eNOS-KO and wild-type (WT) mice. Basal tone and magnitude of FID of arterioles were comparable in the two strains of mice. A role for COX isoforms in the mediation of the responses was assessed by use of valeryl salicylate (3 mM) and NS-398 (10 microM), inhibitors of COX-1 and COX-2, respectively. In eNOS-KO arterioles, valeryl salicylate or NS-398 alone inhibited FID (at maximal flow rate) by approximately 51% and approximately 58%, respectively. Administration of both inhibitors eliminated the dilation. In WT arterioles, inhibition of COX-2 did not significantly affect FID, whereas inhibition of COX-1 decreased the dilation by approximately 57%. The residual portion of the response was abolished by additional administration of Nomega-nitro-L-arginine methyl ester. Western blot analysis indicated a comparable content of COX-1 protein in arterioles of WT and eNOS-KO mice. COX-2 protein, which was not detectable in arterioles of WT mice, was strongly expressed in arterioles of eNOS-KO mice, together with an upregulation of COX-2 gene expression. Immunohistochemical staining confirmed the presence of COX-2 in the endothelium of eNOS-KO arterioles. In conclusion, COX-2-derived PGs are the mediators responsible for maintenance of FID in arterioles of eNOS-deficient mice.

EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice.

Vasodilation to increases in flow was studied in isolated gracilis muscle arterioles of female endothelial nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT) mice. Dilation to flow (0-10 microl/min) was similar in the two groups, yet calculated wall shear stress was significantly greater in arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin, which inhibited flow-induced dilation in vessels of WT mice by approximately 40%, did not affect the responses of eNOS-KO mice, whereas miconazole and 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses. Basal release of epoxyeicosatrienonic acids from arterioles was inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in arterioles of eNOS-KO mice but had no effect on arterioles of WT mice. In WT mice, neither N(omega)-nitro-L-arginine methyl ester nor miconazole alone affected flow-induced dilation. Combination of both inhibitors inhibited the responses by approximately 50%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone inhibited flow-induced dilation by approximately 49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT mice, nitric oxide and prostaglandins mediate flow-induced dilation. When eNOS is inhibited, endothelium-derived hyperpolarizing factor substitutes for nitric oxide. In female eNOS-KO mice, metabolites of cytochrome P-450, via activation of large-conductance Ca2+-activated K+ channels of smooth muscle, mediate entirely the arteriolar dilation to flow.

Gender-specific compensation for the lack of NO in the mediation of flow-induced arteriolar dilation.

Flow-induced dilation of gracilis muscle arterioles was examined in both genders of control rats and rats chronically treated with N(omega)-nitro-L-arginine methyl ester (L-NAME). After L-NAME treatment (4 wk), systolic blood pressure was significantly increased compared with control, whereas the plasma concentration of nitrate/nitrite was significantly reduced. Isolated and pressurized arterioles dilated significantly in response to increases in flow (0-25 microl/min). Flow-induced dilation was comparable in arterioles of control and L-NAME-treated rats but was significantly greater in female than in male rats. L-NAME + indomethacin, which abolished flow-induced dilation in arterioles of male control rats, inhibited the dilation by only ~75% in female control rats. The residual portion of the response was eliminated by additional administration of miconazole, an inhibitor of cytochrome P-450. Indomethacin did not affect the dilation in female L-NAME-treated rats but completely inhibited the response in male L-NAME-treated rats. The indomethacin-insensitive, flow-induced dilation in female L-NAME-treated arterioles was abolished by miconazole, 6-(2-proparglyoxyphenyl)hexanoic acid, or charybdotoxin. Thus an augmented release of endothelial prostaglandins accounts for the preserved flow-induced dilation in arterioles of male rats, whereas a metabolite of cytochrome P-450 is responsible for the maintenance of flow-induced dilation in female rats, suggesting important differences in the adaptation of the endothelium of arterioles from male and female rats to the lack of nitric oxide (NO) synthesis.

Enhanced release of prostaglandins contributes to flow-induced arteriolar dilation in eNOS knockout mice.

Nitric oxide and prostaglandins were shown to contribute to the endothelial mediation of flow-induced dilation of skeletal muscle arterioles of rats. Thus, we hypothesized that flow-induced dilation and its mediation are altered in gracilis muscle arterioles of mice deficient in the gene for endothelial nitric oxide synthase (eNOS-KO) compared with control wild-type (WT) mice. Gracilis muscle arterioles ( approximately 80 micrometer) of male mice were isolated, then cannulated and pressurized in a vessel chamber. The increases in diameter elicited by increases in perfusate flow from 0 to 10 microq/min were similar in arterioles from eNOS-KO (n=28) and WT (n=22) mice ( approximately 20 micrometer at 10 microL/min flow). Removal of the endothelium eliminated flow-induced dilations in vessels of both strains of mice. N(omega)-nitro-L-arginine (L-NNA, 10(-4) mol/L) significantly inhibited flow-induced dilation in arterioles of WT mice by approximately 51% but had no effect on responses of arterioles from eNOS-KO mice. Indomethacin (INDO, 10(-5) mol/L) inhibited flow-induced dilation of WT mice by approximately 49%, whereas it completely abolished this response in arterioles of eNOS-KO mice. Simultaneous administration of INDO and L-NNA eliminated flow-induced responses in arterioles of WT mice. Dilations to carbaprostacyclin were similar at concentrations of 10(-8) and 3x10(-8) mol/L but decreased significantly at 10(-7) mol/L in arterioles of eNOS-KO compared with those of WT mice. These findings demonstrate that, despite the lack of nitric oxide mediation, flow-induced dilation is close to normal in arterioles of eNOS-KO mice because of an enhanced release of endothelial dilator prostaglandins and suggest that this vascular adaptation may contribute to the regulation of peripheral resistance in eNOS-KO mice.

Attenuation of blood flow-induced dilation in arterioles after muscle contraction

The response of third-order arterioles (n = 15) in rat cremaster muscle to increased luminal flow was studied after brief (20-30 s) occlusion of a neighboring arteriole in pentobarbital-anesthetized rats. Red cell velocity increased almost fivefold (485 +/- 54% of control) during occlusion, and vessel diameter increased 63 +/- 11%. Initially, the calculated wall shear rate increased to 430 +/- 40% of control during occlusion but then decreased to 308 +/- 35% of control as a consequence of arteriolar dilation. The muscle was subsequently stimulated to contract for 1 min, and the occlusion procedure was repeated after arteriolar diameter and red cell velocity had returned to control levels. In this instance the vessel dilation was 34 +/- 10% or about one-half of that seen during the previous occlusion, although velocity and shear rate rose to a similar degree (474 +/- 54 and 397 +/- 35%, respectively). Dilation during a third occlusion 2-7 min after the vessel recovered from the second occlusion was as great as during the first occlusion (77 +/- 20%). The results indicate that flow-induced dilation in arterioles of rat cremaster muscle is transiently attenuated after muscle contraction.

Impaired nitric oxide-mediated flow-induced dilation in arterioles of spontaneously hypertensive rats.

We tested the hypothesis that impairment of flow-dependent dilator mechanisms of skeletal muscle arterioles is one of the underlying reasons for the increased peripheral resistance in hypertension. Isolated, cannulated arterioles (approximately 55 microns) of gracilis muscle of 12-week-old spontaneously hypertensive (SH) and normotensive Wistar (NW) rats were investigated. At a constant perfusion pressure (80 mm Hg), the active diameters of NW and SH arterioles were 57.7 +/- 1.9 and 51.5 +/- 3.2 microns, whereas their passive diameters (Ca(2+)-free solution) were 113.6 +/- 2.9 and 101.7 +/- 2.9 microns, respectively. Flow-induced dilation was elicited by increases in flow of the perfusion solution from 0 to 25 microL/min in 5-microL/min steps. This response was significantly less in arterioles of SH compared with NW rats. For example, at 25-microL/min flow, the diameter of arterioles of SH rats was approximately 56% less (P < .05) than those of NW rats. Indomethacin, an inhibitor of prostaglandin synthesis, significantly attenuated the flow-diameter curve in both strains of rats. In contrast, N omega-nitro-L-arginine, a nitric oxide synthase inhibitor, significantly shifted the flow-diameter curve to the right in NW rats, but it did not affect the flow-diameter curve in SH rats. Thus, the present findings demonstrate that in gracilis muscle arterioles of normotensive rats in response to increases in flow (shear stress), prostaglandins and nitric oxide are co-released, resulting in a dilation. In early hypertension, however, there is a reduced arteriolar dilation to increases in flow that is due to the impairment of the nitric oxide-mediated portion of the flow-dependent arteriolar dilation.