bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosine does not vasodilate skeletal muscle arterioles via secondary nitric oxide or prostaglandin production
Authors: Murrant Coral Lyn , Muckle Fiona , Inch Kristin
Published: June 2021

We have recently shown that ADO is responsible for arteriolar vasodilation in response to skeletal muscle contraction. To determine whether the arteriolar vasodilation induced by adenosine (ADO) is, in part, due to the production of nitric oxide and prostaglandins we stimulated transverse arterioles (approx. 40μm) in a hamster cremaster preparation with a range of concentrations of ADO (10−7–10−5M) and measured the diameter of arterioles in the absence of and in the presence of cyclooxygenase inhibitor indomethacin (10−6M INDO), or nitric oxide synthase inhibitor L‐NAME (10−6M), or both. We further investigated whether there was an inhibition of ADO dilation in the presence of L‐NAME and nitric oxide donor SNAP (10−7M). We observed significant vasodilation at all concentrations of ADO (change in diameter with 10−7M ADO:4.8±1.0μm, 10−6M: 11.5±1.5μm, 5×10−6M:18.9±1.7μm, 10−7M: 22.0±1.5μm) that were not significantly inhibited by INDO or L‐NAME, or INDO plus L‐NAME. Further, supplementation of the L‐NAME inhibition with SNAP did not alter any vasodilation induced by ADO. These findings show that ADO does not cause vasodilation through the production of nitric oxide or prostaglandins. These data indicate that the ADO involved in arteriolar vasodilation in response to muscle contraction is not responsible for producing secondary vasodilators such and nitric oxide or prostaglandins.
Autonomic Impairment During Severe Hemorrhage in Obese Zucker Rats
Authors: Clemmer John S. , Xiang Lusha , Lu Silu , Lee Lynn , Hester Robert L.
Published: June 2021

Obesity is characterized by impaired autonomic activity which may adversely affect cardiovascular responses under pathological conditions such as acute hypovolemia. We hypothesize that obesity can impair acute blood pressure regulation during hemorrhage via lack of sympathetic effectiveness and impaired neural reflexes. Severe hemorrhage (a loss of 35% of total blood volume) was induced via femoral catheter (approximately ~0.7ml/min, 11–12 minutes) in conscious lean (LZ) and obese Zucker rats (OZ) (11–12 weeks). Blood pressures and heart rate were monitored during hemorrhage and during 30 minutes of recovery. Control blood pressures and heart rates were not statistically different between LZ and OZ. During hemorrhage blood pressure in the OZ dropped below basal after a 5% loss (Figure 1), while a decrease in blood pressure was not evident until 15% loss of total blood volume in LZ. An elevation in heart rate was sustained until 25% blood loss in LZ but only until 5% blood loss in the OZ (Figure 2). Blood pressure was partially compensated 30 minutes after the completion of hemorrhage in both LZ and OZ, but the OZ blood pressure was significantly lower than the LZ. During the first 5 minutes after completion of the hemorrhage heart rate was decreased in both OZ and LZ with a significantly greater decrease with OZ. In addition, the heart rate was elevated in the LZ at the end of the 30 minute recovery but remained decreased in OZ (Figure 2). Our results suggest that, in response to acute hemorrhage, the compensatory mechanisms mediated by sympathetic activation such as increase in peripheral resistance and tachycardia are blunted in OZ, resulting in increased vulnerability to volume loss and impaired recovery.

Supported by NIH HL‐51971 and HL‐89581.
Capillaries are Embedded in the Sarcolemma of Murine Slow Twitch Skeletal Muscle Fibers
Authors: Glancy Brian , Hsu Li-Yueh , Dao Lam , Bakalar Matthew , French Stephanie , Chess David J , Taylor Joni L , Daniels Mathew P , Esfahani Shervin , Balaban Robert S
Published: June 2021

The purpose of this study was to evaluate the in vivo, 3D spatial relationship between capillaries (CAP), mitochondria (MITO), and three fiber types within murine Tibialis anterior (TA) muscle. Two photon excitation microscopy was utilized for in vivo imaging of MITO (NADH autofluorescence), vasculature, and interstitial space using injected dyes. A custom program was used to trace fibers and determine contact and volume ratios between CAP and 127 muscle fibers. Fiber type (slow (ST), intermediate (IT), fast‐twitch (FT)) was determined from relative NADH intensity and fiber size. Microscopy revealed that half of ST fiber CAP (53.2 ± 4.0%) had at least 50% of their circumference embedded in a groove in the sarcolemma. Embedded CAP were tightly associated with dense MITO populations lateral to the CAP grooves but nearly absent below the groove where fibrils dominated. CAP embedding was significantly lower in IT (29.5 ± 4.2%) and FT (14.2 ± 1.8%). Electron microscopy confirmed ST embedded CAP and MITO localization. A specialized sarcolemma/CAP groove is present in murine ST fibers resulting in CAP embedding in a MITO rich region. This structure optimizes CAP O2 delivery to highly aerobic ST fibers and selectively restricts O2 delivery to FT fibers. Localized contact of CAP with ST plasma membranes suggests receptors or transporters for blood borne signaling or substrate molecules may also be concentrated in these regions.
RhoA/ROCK‐dependent moesin phosphorylation regulates AGEe‐induced endothelial response
Authors: Huang Qiaobing , Wang Jiping , Liu Hongxia , Chen Bo , Li Qiang , Huang Xuliang
Published: June 2021

To investigate the involvement of RhoA/ROCK‐dependent moesin phosphorylation in endothelia stimulated by advanced glycation end products (AGEs), human dermal microvascular endothelial cells (HMVECs) were used to observe the monolayer permeability and distribution of F‐actin. Activations of RhoA and ROCK were determined by a luminescence‐based assay and immunoblotting. RhoA N19 or RhoA L63 was transfected to down‐regulate RhoA expression or over‐express RhoA in HMVECs. H‐1152 was employed to block activation of ROCK. Co‐immunoprecipitation was used to confirm the interaction of ROCK and moesin. To identify AGE/ROCK‐induced phosphorylation site in moesin, mutant pcDNA3/HA‐moesinT558A or pcDNA3/HA‐moesinT558D were applied in endothelia. The results showed that AGE‐HSA increased the monolayer permeability and triggered the formation of F‐actin stress fibers. AGE‐HSA enhanced RhoA/ROCK in a time‐ and dose‐dependent manner. RhoA N19 or H‐1152 abolished these AGE‐induced changes, while RhoA L63 reproduced the AGE‐evoked responses. It is confirmed that AGE induced direct interaction of ROCK and moesin. Thr558 was identified as the phosphorylating site for moesin in AGE‐evoked endothelia. These results indicate the effects of RhoA/ROCK pathway and subsequent moesin phosphorylation in AGE‐mediated endothelial dysfunction. (supported by NSFC30771028, 30971201; IRT0730 and G2005CB522601)
Mesenchymal stem cells isolated from bone marrow and compact bone express both VEGF and PLGF
Authors: Varshney Rohan R. , Lloyd Pamela G.
Published: June 2021

Chronic obstructive pulmonary disease (COPD) is associated with dysregulation of vascular endothelial growth factor (VEGF) family protein levels (decreased VEGF and/or increased placental growth factor, PLGF). We hypothesized that mesenchymal stem cells (MSC) could enhance maintenance and repair of lung vasculature in COPD through VEGF family signaling. Toward this goal, we characterized VEGF and PLGF expression in MSC isolated from mouse bone marrow and compact bone. MSC were cultured in 5% O2, 10% CO2 and 85% N2 and characterized by flow cytometry. MSC from both sources highly expressed the MSC marker CD44 and moderately expressed MSC markers CD105 and CD73. MSC were cultured up to passage 2. Media was collected from primary MSC and p1–2 MSC for ELISA of VEGF and PLGF expression. Fresh media (DMEM + 15% FBS) contained neither PLGF nor VEGF. ELISA of conditioned media from MSC showed that MSC express both VEGF and PLGF, with VEGF>PLGF. VEGF expression increased with passage number, whereas PLGF expression decreased with passage number. Pericytes, cells found in association with the vascular wall, are very similar structurally and functionally to MSC. MSC have also been reported to share similarity with vascular smooth muscle cells (SMC). Our findings suggest that MSCs have a SMC‐like pattern of VEGF and PLGF expression, consistent with a role in vascular maintenance and repair. Funding: OCASCR, NIH R01 HL084494 (PL).
Mechanical ventilation induces a time‐dependent reduction in microvascular oxygenation and vascular conductance in the diaphragm
Authors: Davis Robert Thomas , Bruells Christian S , Stabley John N , McCullough Danielle J , Powers Scott K , Behnke Bradley J
Published: June 2021

Prolonged mechanical ventilation (MV) promotes diaphragmatic atrophy and contractile dysfunction, collectively termed ventilator‐induced diaphragmatic dysfunction. A local mismatching of O2 delivery (QO2) to consumption (VO2) within the diaphragm may contribute, in part, to muscle dysfunction and weaning difficulties following MV. We tested the hypotheses that longer durations of MV (i.e., 6 hrs.) will 1) cause a mismatching of QO2‐to‐VO2, evident through a reduction microvascular PO2 (i.e., PO2m); and 2) reduce vascular conductance (VC) compared to acute MV (i.e., 30 min). Diaphragm PO2m and VC were measured in vivo in adult female Sprague‐Dawley rats (n=15). Data was collected during spontaneous breathing (SB) and after acute MV and 6 hr of MV. PO2m in the diaphragm was significantly reduced following the onset of MV (SB: 53±6, acute MV: 37±8 mmHg; P<0.05) and by an additional ~51% after 6 hr (versus acute MV) to 18±5 mmHg (P<0.05). Diaphragm VC was diminished after the onset of MV (SB: 0.36±0.02, acute MV: 0.24±0.02) and reduced further after 6 hr MV (0.08±0.01 ml/min/100 g/mmHg; all P<0.05). The diminished microvascular driving pressure of O2 and VC provides important insight, and a potential contributing mechanism, into the inadequate maintenance of the QO2‐to‐VO2 ratio in the diaphragm with prolonged MV. The mechanisms responsible for the reduced PO2m and VC with MV require further studies. (NIH and FBRP)
Age‐Related Changes in the Expression of Elastin in Small cerebral and Mesenteric Arteries
Authors: Nourian Zahra , Li Min , Clifford Philip S. , Stupica Aaron , Krenz Maike , Baines Chris , Meininger Gerald A. , Hill Michael A.
Published: June 2021

Recent studies showed an intricate arrangement of elastin fibers in the walls of small arteries as well as marked differences in elastin content and distribution of cerebral vessels compared to those of mesentery or cremaster muscle. The present studies extended these observations to examine age‐related changes in elastin mRNA expression. Total RNA was extracted from small cerebral and mesenteric arteries of Sprague‐Dawley rats aged 3, 7, 11, 19 days, 2 mths and 2 yrs. Elastin mRNA expression levels were determined using qPCR. Relative expression levels were quantified using the 2−ΔΔCt method. Elastin mRNA levels for both vessel types increased from day 3 to a peak at 11 days after which levels declined throughout life. Peak levels of elastin mRNA expression were significantly greater in mesenteric compared to cerebral vessels (relative expression 24.23±2.90 and 14.78±1.23) and remained elevated at later time‐points. Differences in mRNA expression between cerebral and mesenteric vessels were consistent with earlier observations showing more elastin protein in the adventitia and media of mesenteric arteries. In addition to the temporal pattern of mRNA expression, the data show heterogeneity in magnitude of elastin expression in resistance artery walls from differing vascular beds. Such differences may impact function through their effects on the mechanical environment of the vessel wall. (Supp NIH HL095486)
Placenta growth factor is regulated by hydrogen peroxide at the post‐transcriptional, but not the transcriptional, level in vascular smooth muscle
Authors: Shaw Jennifer H , Lloyd Pamela G
Published: June 2021

When supply arteries are occluded, arteriogenesis occurs resulting in the expansion of collateral arteries. Placenta growth factor (PLGF), a VEGF‐family protein, is a mediator of arteriogenesis, but little is known about its regulation. We previously showed that hydrogen peroxide (H2O2) is a key paracrine regulator of PLGF mRNA in vascular smooth muscle cells. We also determined that H2O2 regulates PLGF at the post‐transcriptional level in human coronary artery smooth muscle cells (CASMC) by significantly extending the half‐life of PLGF mRNA and this regulation requires p38 MAPK, JNK and ERK1/2 pathways. Pro‐inflammatory stimuli have been shown to increase PLGF promoter activity in other cell types and the PLGF promoter contains binding sites for transcription factors responsive to oxidative stress. Therefore, we hypothesized that H2O2 exerts transcriptional control of PLGF mRNA in CASMC in addition to post‐transcriptional regulation. To test our hypothesis, CASMC were co‐transfected via electroporation with a vector containing the PLGF promoter sequence linked to a firefly luciferase reporter gene and the Renilla luciferase vector as an internal control for transfection efficiency. Co‐transfected CASMC treated with a single dose of H2O2 (50 μM) showed no significant difference from basal PLGF promoter activity observed in untreated co‐transfected CASMC (n=3). We conclude that H2O2 does not regulate PLGF at the transcriptional level in CASMC.
Nitric Oxide Synthase Inhibition Weakens Beneficial Effects of Increased Plasma Viscosity on Cardiac Function in Hemodilution Model
Authors: Chatpun Surapong , Cabrales Pedro , Tsai Amy , Intaglietta Marcos
Published: June 2021

This study tested the hypothesis that the inhibition of nitric oxide synthase negatively affects the beneficial effects of high viscogenic plasma expander (HVPE) on cardiac performance in a state of hemodilution. Five anesthetized hamsters were administered with N(G)‐nitro‐L‐arginine methyl ester (L‐NAME), nitric oxide synthase inhibitor, as a tested group whereas other five without L‐NAME infusion were used as a control group. Animals were performed an isovolemic hemodilution by 40% of estimated blood volume with dextran 2000 kDa, HVPE with viscosity 6.34 cP. L‐NAME administration followed by isovolemic hemodilution significantly decreased heart rate from 441Â±41 bpm at baseline to 355Â±27 bpm at 60 min after hemodilution. End‐systolic pressure increased 14% after L‐NAME infusion and maintained higher than baseline after hemodilution whereas it gradually decreased in animals without L‐NAME infusion. L‐NAME significantly decreased the maximum rate of ventricular pressure change, stroke volume and cardiac output after hemodilution compared to a control group. Systemic vascular resistance increased to 162% and 135% of baseline after L‐NAME infusion and 60 min after hemodilution which is particularly higher than a control group. These results indicate that nitric oxide induced by HVPE play a major role on cardiac function during an isovolemic hemodilution.
Membrane acid‐base transporters modulate artery structure
Authors: Boedtkjer Ebbe , Bentzon Jacob , Damkier Helle , Füchtbauer Ernst-Martin , Falk Erling , Aalkjaer Christian
Published: June 2021

Acid‐base transport and pH control modulate cell proliferation and migration with possible consequences for artery remodeling. Here, we used knockout mice for the Na+/H+‐exchanger NHE1 and Na+,HCO3−‐cotransporter NBCn1 to study their role in determining arterial structure. Measurements were performed using pH‐sensitive fluorophores, myography and stereological analyses. NHE1 mediated the Na+/H+‐exchange in mesenteric arteries, and knockout of NHE1 was associated with reduced media thickness and an equivalent inhibition of maximum active tension development. The reduced media thickness was due to a smaller smooth muscle cell volume and cross‐sectional area with no change in the number of smooth muscle cells per unit artery length. NBCn1 mediated the Na+,HCO3−‐cotransport in carotid arteries. Following partial carotid artery ligation, the inward remodeling (reduction in carotid artery diameter and increase in media area) in wild type mice was more than 50% attenuated in NBCn1 knockout mice. In conclusion, we show that the acid‐base transport proteins NBCn1 and NHE1 play an important role for determining artery structure. Supported by Danish Council for Independent Research and Danish Heart Foundation.
Estimating peripheral blood pressure from central blood pressure by a transfer function method
Authors: Rose William C. , Schell Kathleen A. , DiSabatino Angela , Stillabower Michael E.
Published: June 2021

Objective

Determine whether blood pressure measured by a cuff on the forearm could be predicted better by upper arm cuff pressure or by radial pressure estimated by a transfer function method from the ascending aortic pressure wave.

Subjects

32 adults undergoing cardiac catheterization.

Methods

Blood pressure was measured by cuffs on the forearm and upper arm and by a catheter in the ascending aorta. A transfer function was used to predict radial artery blood pressure from the ascending aortic pressure waveform. Regressions were computed between upper arm pressure measured by cuff and forearm pressure measured by cuff, and between predicted radial artery pressure and forearm pressure measured by cuff.

Results

The transfer function method did not predict forearm systolic pressure as well as the upper arm cuff did (r=0.76 for the former, r=0.89 for the latter). For diastolic pressure and mean pressure, the transfer function method predicted slightly less well than the upper arm cuff (r=0.83 vs. r=0.87 for diastolic; r=0.77 vs. r=0.80 for mean).

Conclusions

The transfer function method does not predict forearm cuff blood pressure better than an upper arm cuff measurement. Supported by the University of Delaware Research Foundation.
Deletion of protein tyrosine phosphatase 1B (PTP1B) prevents type 1 diabetes‐induced vascular dysfunction
Authors: Norman Joseph Blake , Herren David , Tremblay Michel L , Stepp David W , de Chantemele Eric J Belin
Published: June 2021

Impaired vascular function is a common feature in type I diabetic patients. PTP1B deletion prevents endothelial dysfunction and leptin infusion restores cardiovascular dysfunction induced by type I diabetes. We hypothesized that increasing leptin sensitivity via PTP1B deletion prevents vascular dysfunction in type I diabetic mice. Type I diabetes was induced with streptozotocin (STZ) in male and female Balb/C and PTP1B KO mice. Vascular function was studied after 24 days of STZ on aortic rings mounted on a wire myograph. Glycemia was significantly increased with STZ and further exaggerated by PTP1B deletion in male mice only (Balb/CMSTZ: 342±18 vs PTP1BMSTZ: 509±26 mg/dl, p<0.05). Endothelial function assessed via acetylcholine (ACh)‐mediated relaxation was reduced in both male and female Balb/C mice but preserved in PTP1B KO animals. While LNAME completely abolished ACh‐mediated relaxation in Balb/C mice, addition of indomethacin was required to abolish relaxation in PTP1B KO mice treated with STZ, suggesting dilatory cyclooxygenase derivatives involvement. Phenylephrine (PE)‐induced constriction was increased by STZ in male and female Balb/C, but preserved in PTP1B KO male mice only. Taken together these data indicate that PTP1B deletion protects against the harmful vascular effects of type I diabetes, independent of hyperglycemia and with more pronounced effects in male than female mice.
Bicarbonate transport drives Na‐independent K secretion (NIKS) via BK‐α/β4 in β‐intercalated cells (IC‐β)
Authors: Cornelius Ryan J , Hatcher Lori I , Echtenkamp Kari M , Contreras Jacob I , Sansom Steve C
Published: June 2021

The Yanomamo tribe, who exemplify NIKS, consume a low Na, high K, alkaline diet and excrete a high volume of urine (control = 9.2 ml/kg/day; Yanomami = 20.7 ml/kg/day) with a low urinary [Cl] (15 mM) and [Na]/[K] (0.007). We hypothesized that the mechanism for NIKS involves KHCO3 secretion via BK‐α/β4 in beta intercalated cells (IC‐β). To test this hypothesis, we fed WT mice (7 d) a low Na (0.01%), high K (5%), alkaline diet (LNaHK). The urinary [Na]/[K] of LNaHK was 0.009 ±0.001, similar to Yanomami. Immunohistochemical analysis of LNaHK kidneys revealed increased V‐ATPase and proportion of IC‐β. The urine pH was 6.3 in control and 8.6 in LNaHK, consistent with the low urinary [Cl] of Yanomami. Mice exhibited a larger increased urinary flow (control = 53 ±5 ml/kg/day, LNaHK = 242 ±19 ml/kg/day), compared with Yanomami. Using this model, we determined whether a knockout of the BK‐β4 (β4KO) exhibited a defect in NIKS. The urinary flow of β4KO on LNaHK was 195 ±17 ml/kg/day, a value slightly lower than WT; however, the trans‐tubular K gradient (TTKG), a measure of Vte‐driven lumen to plasma [K] in the terminal cortical collecting ducts, was 27.5 ±2.0 in WT but reduced to 15.5 ±1.2 in β4KO. When WT were given NH4Cl water with LNaHK diet, the TTKG was reduced to 16.4 ±1.4. Summary: 1. The LNaHK mouse is a good model for NIKS. 2. The activity of BK‐α/β4 is necessary for NIKS. 3. IC‐β activity is enhanced in NIKS. 4. NIKS is inhibited by an acid load.
Interactions of Polycystin 2 and TRPV4 in Polycystic Kidney Disease
Authors: Sas Kelli Margot , Amria May , Bell Phillip Darwin
Published: June 2021

Transient receptor potential (TRP) channels are cation‐selective ion channels that have been implicated in polycystic kidney disease (PKD). Mutations in the TRP protein polycystin 1 (PC1) and polycystin 2 (PC2), which is a calcium permeable channel, cause autosomal dominant PKD. Recently, we have found that PC2 and TRPV4 form a 23 pS multimeric calcium‐permeable channel in cultured renal collecting duct cells. Previous studies have evaluated the role of PC2 in PKD but have not considered whether TRPV4 might influence cystic development. The goal of the current studies was to determine the in vivo significance of the PC2\TRPV4 channel complex in PKD, with the hypothesis that simultaneous knockdown of PC2 and TRPV4 might reduce cystic burden. We utilized mice with a conditional floxed allele for PC1 or PC2 and mated them with a control mouse or a mouse that had a global conventional knockdown of TRPV4. PC1 and PC2 were knocked out in adult mice by tamoxifen administration followed by nephrectomy, which we have previously shown accelerates cystogenesis in the adult mouse. After three months, histological sections were examined from the remaining kidney for overall cystic burden. Knockout of PC1or PC2 alone or both PC1 and TRPV4 resulted in very large cystic kidneys. With deletion of both PC2 and TRPV4 kidney size was relatively normal and there was substantially less cysts compared to the other groups. We conclude that deletion of both PC2 and TRPV4 reduces cystogensis presumably by the elimination of the 23 pS calcium entry pathway. NIH & VA
Amelioration of insulin resistance by rosiglitazone is associated with increased adipose cell size in obese type 2 diabetics
Authors: Yang Jian , Eliasson Bjorn , Smith Ulf , Cushman Samuel W. , Sherman Arthur
Published: June 2021

Whereas early studies reported that having large adipose cells correlates with insulin resistance, this is not always seen. A recent study with BMI‐matched moderately insulin‐sensitive or –resistant obese subjects found a correlation with the proportion of small adipose cells, rather than the size of the large adipose cells, suggesting impaired adipogenesis. To further test this hypothesis, we recruited and treated eleven type 2 diabetic patients for 90 days with an insulin‐sensitizing drug rosiglitazone. Needle biopsies of the abdominal subcutaneous fat from the patients were assayed by fitting the adipose cell size distribution with two exponentials and a Gaussian function. The fraction of large adipose cells was defined as the area of the Gaussian peak and the size of the large cells was defined as its center. Insulin resistance was ameliorated as shown by lowered fasting plasma glucose and increased plasma adiponectin. Rosiglitazone also increased mRNA expression of Adiponectin and GLUT4 in adipose tissue. We observed multimodal adipose cell size distributions similar to those seen previously in non‐diabetic, moderately obese subjects. Contrary to expectations, the main effect of rosiglitazone was increased size of the large adipose cells. We conclude that improved insulin sensitivity is associated with increased size of large adipose cells in rosiglitazone‐treated type 2 diabetics.
Impaired Fasting Glucose and Enhanced Endothelin‐1 Vasoconstrictor Tone
Authors: Ma Julie , Diehl Kyle , Templeton Danielle , Greiner Jared , Stauffer Brian , DeSouza Christopher
Published: June 2021

Impaired fasting blood glucose (IFG: 100–125 mg/dL) is a risk factor for type 2 diabetes, hypertension and cardiovascular disease (CVD). Endothelin‐1 (ET‐1) is a potent vasoconstrictor peptide produced by the vascular endothelium. ET‐1 plays a central role in the regulation of vasomotor tone and contributes etiologically to the development of hypertension and CVD. ET‐1‐mediated vasoconstrictor tone has been shown to be elevated in adults with type 2 diabetes. Whether ET‐1 vasoconstrictor tone is also elevated in adults with IFG is unknown. The aim of this study was to determine the influence of impaired fasting glucose on ET‐1‐mediated vasoconstrictor tone. Forearm blood flow (FBF: plethysmography) responses to intra‐arterial infusion of selective ETA receptor blockade (BQ‐123: 100 nmol/min for 60 min) and non‐selective ETA/B blockade (BQ‐123 + BQ‐788, 50 nmol/min for 60 min) were determined in 28 adults (17 M/11 F): 14 normal fasting glucose (NFG; age: 57±2 yr; BMI: 29.2±0.9 kg/m2; glucose: 88±2 mg/dL) and 14 IFG (58±1; 29.6±1.1 kg/m2; 105±1 mg/dL). BQ‐123 elicited a significantly greater increase in FBF in the IFG (~30%) than NFG (~10%) adults. Addition of BQ‐788 resulted in a further increase (~20%; P<0.05) in FBF in IFG but not NFG adults. ET‐1‐mediated vasoconstrictor tone is elevated with IFG. Enhanced ET‐1 vasoconstrictor tone may underlie the increased risk of hypertension and CVD in adults with IFG.
Role of miRNAs for vascular smooth muscle mechano‐sensing and contractile function
Authors: Albinsson Sebastian
Published: June 2021

The phenotype of smooth muscle cells is orchestrated by regulation of mRNA and protein expression, which is partly controlled by mechanical stimuli. miRNAs are small non‐coding RNAs that participate in the regulation of mRNA and protein expression and are now known to be involved in smooth muscle differentiation and growth.

The objective of this study was to investigate the role of miRNAs for smooth muscle mechano‐sensing and contractile function.

The importance of miRNAs was identified by stretch or pressurization of vessels from Dicer KO and control mice. Dicer is an essential for the formation of most mature miRNAs. We found that stretch‐sensitive contractile differentiation was reduced in Dicer KO portal veins. Furthermore, myogenic tone and pressure induced signalling events were abolished in pressurized arteries from Dicer KO mice. Finally, we found a reduced expression of L‐type calcium channels in Dicer KO vessels, which may be an important mechanism behind these effects.

In conclusion, miRNAs play an important role for vascular mechano‐sensing and contractile function.

This study is primarily funded by the Swedish Heart and Lung Foundation and the Crafoord Foundation
Integrin‐dependent and ‐independent potentiation of BKCa channel current by cell stretch
Authors: Gui Peichun , Braun Andrew P , Davis Michael J
Published: June 2021

The large conductance, calcium‐activated K+(BKCa) channel is an important regulator of stretch‐induced contraction of VSM. Integrins are involved in this process because α5β1 and αvβ3 integrin blocking antibodies prevent myogenic constriction. Recent studies in our lab indicate that fibronectin (FN), an endogenous α5β1 integrin ligand, enhances BKCa channel current through both Ca2+‐ and phosphorylation‐dependent mechanisms in native VSMCs or HEK293 cells transfected with BKCa cDNA. To test whether BKCa channels are mechanosensitive and the possible role of integrins in this process, patch clamp methods were used to investigate the properties of either native or heterologously expressed BKCa channels by stretch of single cells plated onto a flexible substrate. Thin silicone membranes were coated with either FN or poly‐l‐lysine (PLL) to assess integrin‐dependent and ‐ independent responses, respectively, and stretched using two blunt micropipettes driven in equal and opposite directions by piezoelectric translators. Graded stretch to 130% of resting cell length induced graded increases in BKCa current (up to 95%) in HEK 293 cells expressing murine BKCa channel α‐subunits (mSlo). No potentiation was evident in mock transfected cells (n=10). The increase in current was ~2‐fold greater for cells adhering to FN (n=9) than for cells on PLL (n=7). On FN, 130% longitudinal stretch of primary VSM cells (n=3) caused ~2x potentiation of the basal outward current, and ~3x potentiation of the iberiotoxin‐sensitive current. Our results suggest that the BKCa channel can be potentiated by membrane stretch, with one component due to intrinsic mechanosensitivity of the channel and a second component due to signaling through an integrin‐dependent process.
Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation
Authors: Liang Guo Hua , Xi Qi , Leffler Charles W. , Jaggar Jonathan H.
Published: June 2021

Hydrogen sulfide (H2S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H2S induce vasodilation are unclear. We tested the hypothesis that H2S dilates piglet cerebral arterioles by modulating local and global intracellular Ca2+ signals in smooth muscle cells. High‐speed confocal imaging revealed that H2S increased Ca2+ spark frequency ~1.43 fold and decreased global intracellular Ca2+ concentration ([Ca2+]i) by ~37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H2S did not alter Ca2+ wave frequency. In voltage‐clamped (–40 mV) cells, H2S increased the frequency of Ca2+ spark‐induced transient Ca2+‐activated K+ (KCa) currents ~1.8 fold, but did not alter the amplitude of these events. H2S did not alter the activity of single KCa channels recorded in the absence of Ca2+ sparks in arteriole smooth muscle cells. H2S increased SR Ca2+ load ([Ca2+]SR), measured as caffeine (10 mM)‐induced [Ca2+]i transients, ~1.5 fold. H2S dilated pressurized cerebral arterioles. Iberiotoxin, a KCa channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H2S‐induced vasodilation by ~38 and ~37%, respectively. In summary, our data indicate that H2S elevates [Ca2+]SR, leading to Ca2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient KCa current frequency leads to a reduction in global [Ca2+]i and vasodilation.

NIH/NHLBI
Phenylephrine stimulates the production of reactive oxygen species (ROS) in endothelial cells from hypertensive but not in normotensive rat aorta
Authors: Silva Bruno R , Pernomian Laena , Bendhack Lusiane Maria
Published: June 2021

The endothelial cells (EC) play important role in the vascular tone control. The production of ROS is increased in hypertension that can modify the contractile response to agonists. This study aimed to evaluate the role of EC on the phenylephrine (PE)‐induced contraction in normotensive (2K) and renal hypertensive (2K‐1C) rat aorta and the effect of PE on the production of ROS in EC from both rat groups. Concentration‐effect curves were constructed for PE in 2K and 2K‐1C intact endothelium (E+) or denuded aortas (E−). The efficacy (Emax) and potency (pD2) were compared. Isolated EC were incubated with the ROS‐sensitive dye DHE and the fluorescence intensity (FI) was measured by flow citometry. EC were stimulated with PE (0.1μM) for 10 min with the antioxidants superoxide scavenger Tiron or Catalase. The contraction was similar in 2K and 2K‐1C E− aortas. In E+ aortas the Emax was lower in 2K‐1C rings (1.2±0.2g, n=7 p<0.001) than in 2K (2.2±0.1g, n=5), but the potency was not different between them. Basal FI of DHE on EC was similar in 2K (43388.5±869.8, n=4) and 2K‐1C (37373.0±536.2, n=4), which was increased by PE only in 2K‐1C (68911.5±4146.3, n=4 p<0.001). The enhancement of ROS was reversed to the basal levels by the antioxidants. Our results suggest that the lower contraction induced by PE in intact endothelium 2K‐1C aorta can be due to the EC increased production of ROS stimulated by PE.

Supported by FAPESP, CAPES and CNPq.
Pharmacological studies of BK and L‐type Ca2+ channel function in mesenteric arteries and veins from obese patients
Authors: Xu Hui , Watson Ralph E. , Yenumula Panduranga , Fernandes Roxanne , Pereira-Hicks Cristiane N , Fink Gregory D , Galligan James J
Published: June 2021

Small mesenteric veins (MV) are important in regulation of blood pressure via their capacitance function, however, our previous studies showed that MV displayed heterogeneity in pharmacological responses across species. For example, the large conductance Ca2+ activated K+ (BK) channels and L‐type Ca2+ channels control basal tone and spontaneous oscillations in tone in rat but not murine MV. It is unknown if BK and L‐type Ca2+ channels function in human MV. Therefore, we tested BK and L‐type Ca2+ channel function in isolated and pressurized MA (mesenteric arteries) (60 mmHg) and MV (4 mmHg) from small segments of jejunum harvested from specimens obtained from obese subjects undergoing gastric bypass surgery. The patients reported here were female, 23–45 years old, with BMI values between 34–54, but without hypertension or diabetes. We measured drug‐induced changes in outside diameter and found that: 1) Norepinephrine (NE) dose‐response curves were similar in MA (n=4) and MV (n=4). 2) Nifedipine completely blocked Bay K 8644 (L‐type Ca2+ channel agonist)‐induced MA and MV constriction. 3) Nifedipine also blocked KCl (20–60 mM)‐induced MA, and KCl (20–40 mM)‐induced MV constriction. 4) At physiological intraluminal pressure, MV, but not MA, displayed basal tone and oscillating constrictions which were blocked by nifedipine or removing extracellular Ca2+. 5) Paxilline, a BK channel blocker, caused a 20% constriction in MA. 6) Paxilline did not constrict MV, but completely inhibited spontaneous oscillations in tone. Our results indicate that both BK and L‐type Ca2+ channels regulate human MA and MV tone, but act via blood vessel specific mechanisms. Pharmacological responses of human MV are similar to those of the rat but not the mouse.
Tissue Angiotensin II Type 1 Receptor Expression in Development of Hypertension in SHR/Brown Norway Backcross Rats
Authors: Collett Jason , Ighodaro Adesuwa , Osborn Jeffrey L.
Published: June 2021

Angiotensin II (AII) has been linked to hypertension in Okamoto Spontaneously Hypertensive Rats (SHR). The transmission and tissue specific progeny expression of AII Type 1 receptors (AT1r) in SHR and the development of genetic hypertension remains unknown. It is hypothesized that tissue AT1r expression of genes derived from SHR are linked to elevated blood pressure in offspring of SHR crossed with normotensive Brown Norway rats. A rat colony was developed by breeding a female Okamoto‐Aoki SHR (MAP=160mmHg) with a BN male (MAP=91mmHg). Hypertensive F1 female progeny were backcrossed with the founder BN to generate 5 subsequent backcross generations. F1 and backcross generation 3 (BC3) tissue AT1r protein expression were evaluated. Progeny were grouped as normotensive (NT<105mmHg; F1 n=0, BC4 n=8), borderline hypertension (BHT 105 ≤ MAP < 122 mmHg; F1 n=18, BC4 n=21) and hypertensive (HT MAP ≥ 122 mmHg; F1 n=15, BC4 n=16). AT1r protein expression between HT and BHT F1 progeny were similar in kidney, hypothalamus or forebrain. HT BC3 rats had greater kidney and hypothalamic AT1r expression but not FB compared to NT rats. There was a positive correlation between MAP and AT1r expression in kidney and hypothalami. These results demonstrate that tissue specific elevated AT1r expression may contribute to spontaneous hypertension in SHR/BN rats with minimal SHR genome inheritance.
Increased Myogenic Reactivity and Decreased Vasodilation of Resistance Caliber Uterine Arteries from Pregnant Rats with Periodic Reductions in Uterine Perfusion Pressure
Authors: Reho John J , Toot Jonathan D , Peck Jennifer , Novak Jacqueline , Ramirez Rolando J
Published: June 2021

We have recently demonstrated that periodic reductions in uterine perfusion pressure in pregnant rats results in maternal hypertension and a constrictive phenotype of resistance‐sized mesenteric arteries. The purpose of this study was to examine the effects of periodic reductions in uterine perfusion pressure on uterine artery myogenic reactivity and vasodilation. An aortic occluder was implanted on Day 14 of gestation. The occluder allows uterine perfusion pressure to be reduced by 40% for 1hr a day for 5 consecutive days. Animals were terminated on gestational Day 21 and resistance‐sized uterine arteries were mounted on a pressurized arteriograph where myogenic reactivity and agonist (methacholine) vasodilation were assessed and compared to responses of arteries from SHAM‐operated controls. Myogenic reactivity was increased in resistance‐sized uterine arteries from Occluder animals compared to SHAM (p<0.05). Methacholine dilation of resistance‐sized uterine arteries was reduced in Occluder rats compared to SHAM (p<0.05). These data suggest that Occluder animal uterine artery vasoreactivity is altered toward a more contractile phenotype than SHAM and may play a role in the pathology associated with the model. We speculate that similar changes may have relevance in the pathology of preeclampsia.
Thioredoxin‐1 mediates hypoxia‐induced pulmonary artery smooth muscle cell proliferation
Authors: Tipple Trent E , Nelin Viktoria , Gin Yi , Rogers Lynette , Nelin Leif
Published: June 2021

Pulmonary hypertension (PH) manifests itself in the lungs of both adults and children. The causes of vascular proliferation and remodeling that occur mainly in the pulmonary arterial smooth muscle cells (PASMC) are poorly understood. Hypoxia underlies many forms of PH and alters the redox balance within PASMCs. The aim of this study was to determine the role of the thioredoxin‐ 1 (Trx1) in hypoxia‐induced PASMC proliferation. Human PASMC were exposed to hypoxia (1% O2) or normoxia (21% O2) and Trx1 levels were determined by western blotting. Proliferation studies were done by equally seeding 6 well plates with PASMC, incubating in normoxia or hypoxia for 5 d and counting viable cells using trypan blue exclusion. Our data indicated that hypoxia increases PASMC proliferation. Trx1 levels in hypoxia were significantly greater after 48 and 72 hours of exposure than in PASMC grown in normoxia. Treatment with Trx1‐specific siRNA completely prevented hypoxia‐induced PASMC proliferation. Hypoxia response element (HRE) luciferase reporter studies indicated a 3.5‐fold increase in HRE‐luciferase activity in hypoxia‐exposed PASMCs that was prevented by Trx1 siRNA treatment. Similarly, pharmacological inhibition of Trx1 also prevented hypoxia‐induced increases in PASMC proliferation and HRE‐luciferase activity. Our findings indicate that Trx1 modulates hypoxia‐induced PASMC proliferation, likely through effects on hypoxia‐inducible factor‐mediated pathways. We speculate that Trx1 represents a novel therapeutic target for the vascular remodeling that underlies PH.
Mechanisms that decrease human Neprilysin gene expression in cigarette smoke‐induced pulmonary vascular disease
Authors: Wick Marilee J. , Loomis Zoe L. , Hersh Louis B. , Dempsey Edward C.
Published: June 2021

We previously demonstrated that lung neprilysin (NEP) may be protective against cigarette smoke (CS)‐induced pulmonary vascular remodeling, a major contributor to pulmonary hypertension (PHTN) in lung disorders/diseases, such as chronic obstructive pulmonary disease (COPD). We found decreased NEP levels in COPD lung, at least some of which are due to changes in NEP transcription that may involve epigenetic mechanisms, but not ROS‐induced NEP gene damage or NEP gene promoter methylation. The goal of the present study is to identify CS‐induced mechanisms that decrease NEP gene transcription. We now present evidence that histone proteins may be modified (methylated, deacetylated) and decrease NEP transcription in response to CS. This is shown with human PA SMCs in ChIP experiments with antibodies to modified histone proteins (H3ac, H3K9me2), and with the compounds 5‐Aza‐2′deoxycytidine, Trichostatin A, or Valproic Acid. CS‐specific changes in regulatory or transcription factor (HIF‐1 and 2, NFkB) binding to the NEP gene that could decrease NEP transcription are also investigated in ChIP experiments with PA SMCs. Our results may suggest new ways to increase endogenous pulmonary NEP levels to treat or prevent smoke or hypoxia‐induced pulmonary vascular remodeling.

Support: NHLBI RO3‐HL‐095430, NHLBI PPG P05 HL014985 and VA Merit.
Anti‐endothelial cell antibodies (AECA) in pulmonary arterial hypertension (PAH)
Authors: Morrow Rebekah , Fagan Karen A. , Cioffi Donna L.
Published: June 2021

PAH is a fatal disease that results in right heart failure. PAH pathology is unclear, but endothelial activation and immune responses contribute to vascular remodeling. AECA are found in many autoimmune disorders and both idiopathic and connective tissue disease‐associated PAH. In autoimmune vasculitides, AECA activate endothelial cells through increased reactive oxygen species (ROS) production. While the effects of AECA on endothelial cells have been studied in some diseases, the effects of AECA from PAH on pulmonary endothelial cells are unknown. We sought to determine whether AECA bind and increase ROS in pulmonary endothelial cells.

Methods

Western blots were performed using membrane fractions of human pulmonary and systemic endothelial cells. Blots were treated with serum from PAH patients or healthy controls. Fluorescence microscopy was used to assess the effects of AECA on ROS production in cells treated with IgG from PAH patients or healthy controls.

Results

While some bands were found in both PAH and healthy subjects, a band of 37 kD was seen in pulmonary microvascular endothelial cells when blots were treated with PAH serum. Cells treated with IgG from healthy controls showed no increase in ROS but PAH IgG elicited an increase. These data suggest that some AECA from PAH bind to pulmonary endothelial cells and may lead to ROS generation. Supported by 5R00HL089361 and 5T32HL076125‐8.
The GNAI2 gene ‐ a candidate biomarker of hypertension that determines salt‐resistance vs. salt‐sensitivity in rat models
Authors: Wainford Richard David , Kapusta Daniel R
Published: June 2021

SNP's in the GNAI2 gene increase the hypertension risk in Italian & Japanese populations. We have shown that CNS Gαi2 protein pathways mediate the renal sympathoinhibition and natriuresis evoked by α2‐adrenoceptor activation in‐vivo. Therefore, we examined the role of CNS Gαi2 proteins in the regulation of fluid & electrolyte homeostasis and MAP in response to high salt intake in intact and renal denervated (RDNX) rats. Following 21‐day high salt (HS) intake (8% NaCl diet) we observed a site specific increase in hypothalamic paraventricular nucleus (PVN) Gαi2 proteins in Sprauge‐Dawley (SD) and Dahl Salt‐Resistant (5.8, & 7.6 fold respectively, P<0.05), but not Dahl Salt‐Sensitive (DSS) rats. In SD rats, oligodeoxynucleotide (ODN)‐mediated Gαi2 down‐regulation caused renal nerve‐dependent sodium retention (24h Na+ balance [meq]; Scr + HS 0.3±0.1, Gαi2 + HS 2.9±0.3*, Gαi2 RDNX + HS 0.8±0.4τ), global sympathoexcitation (plasma NE [nmol/L] Scr + HS 49±5, Gαi2 + HS 98±8*, Gαi2 RDNX + HS 73±5τ) and hypertension (MAP [mmHg] Scr + HS 128±3, Gαi2 + HS 147±3*, Gαi2 RDNX + HS 132±2τ). In DSS rats, chronic brain Gαi2 ODN infusion exacerbated salt‐induced hypertension (MAP [mmHg] Scr + HS 164±3 vs. Gαi2 + HS 183±4*), sodium retention and global sympathoexcitation (plasma NE [nmol/L] Scr + HS 87±6 vs. Gαi2 + HS 115±8). In salt‐resistant, but not salt‐sensitive animals, PVN Gαi2 proteins are up‐regulated to mediate endogenous anti‐hypertensive sympathoinhibitory mechanisms to maintain fluid & electrolyte homeostasis and normotension during HS challenge; this may reflect a potential mechanism by which GNAI2 SNP's increase the risk of hypertension in human populations. *p<0.05 vs. Scr + HS, τp<0.05 vs. Gαi2 + HS; HL107330
Role of the sympathetic nervous system in mediating hypertension in a model of polycystic ovary syndrome in rats.
Authors: Lima Roberta , Maranon Rodrigo , Reckelhoff Jane F
Published: June 2021

Hypertension (HT) is one of the major risk factors for cardiovascular disease in women with polycystic ovary syndrome (PCOS). Dysregulation of sympathetic nervous system (SNS) has been proposed to contribute to etiology of HT in women with PCOS. This study tested the hypothesis that activation of the SNS plays an important role in mediating HT in PCOS. We used hyperandrogenomic female (HAF) rat model in which dihydrotestosterone (DHT) (7.5 mg/90 days or placebo (PL)) pellets were implanted subcutaneously in female SD rats at 4 weeks of age and compared to PL. At 8wks age placebo and HAF rats underwent uninephrectomy, after two weeks, underwent unilateral kidney denervation (RD) or sham (SH) surgery and telemetry transmitter implantation. After two weeks recovery (12wks of age, 8wks DHT or PL), mean arterial pressure (MAP) and heart rate (HR) were recorded for 5 days. RD significantly decreased BP in HAF rats compared to SH (104±2mmHg,n=4vs115±3mmHg,n=5;p<0.05), but had no effect on MAP in PL group compared to SH (100±2mmHg vs104±1mmHg, n=5; p>0.05). HR was also not different between PL‐SH compared to RD (398±4mmHg, vs 411±4mmHg, n=5; p>0,05) and HAF‐SH compared to RD (366±10mmHg, n=5 vs 366±6mmHg, n=4; p>0,05). These data indicate that the renal nerves play a key role in the control of MAP in HAF rats, and suggest that elevated MAP in women with PCOS could be treated with SNS blockers. Supported by NIH HL66072, HL69194, HL51971.
Relaxin (RLX) lowers plasma levels of asymmetric dimethylarginine (ADMA) during chronic angiotensin II (ANGII) infusion
Authors: Sasser Jennifer M , Cunningham Mark W , Baylis Chris
Published: June 2021

The endogenous nitric oxide (NO) synthase inhibitor ADMA is increased during chronic ANGII infusion. We and others have shown that RLX attenuates hypertension and increases NO bioavailability. Therefore, this study tested the hypothesis that RLX reduces ADMA levels in ANGII hypertension. Male Sprague‐Dawley rats were given ANGII (400ng/kg/min) or vehicle (CON) for 6 weeks via minipump. RLX was then administered (4 μg/h) to half of the rats for the last 4 weeks (n=6–9 per group). ANGII increased blood pressure vs CON (165±5 vs 104±3 mm Hg, p<0.05), and this was reduced by RLX (135±13, p<0.05 vs both ANGII and CON). ANGII also increased plasma ADMA levels vs CON (1.1±0.2 vs 0.23±0.02 μmol/L, p<0.05), and ADMA levels were normalized by RLX (0.29±0.02 μmol/L, p<0.05 vs ANGII). To determine the mechanisms responsible for the decrease in plasma ADMA, we investigated expression of protein arginine methyl transferase (PRMT) and the expression and activity of dimethylarginine dimethylaminohydrolase (DDAH). PRMT1 abundance was increased in the lung with ANGII vs CON (1.0±0.2 vs 1.5±0.2 units, p<0.05), but was not reduced by RLX (1.7±0.7 units). No changes in PRMT3 in the lung or PRMT1 in the kidney cortex or liver were observed. Furthermore, no changes in DDAH expression or activity were observed in the kidney cortex or liver. Our data do not reflect actions of relaxin in the kidney cortex, liver, or lung to reduce ADMA levels. Relaxin may have effects on other target organs such as by modulating DDAH activity in the vasculature.