bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of dicer in diabetic cardiomyopathy through dysregulation of MMP‐9 and TIMP‐4
Authors: Mishra Paras Kumar , Givvimani Srikanth , Sen Utpal , Abe Oluwasegun A , Tyagi Neetu , Basu Poulami , Munjal Charu , Tyagi Suresh C
Published: June 2021

To test the hypothesis that dysregulation of matrix metalloproteinase‐9 (MMP‐9) and its tissue inhibitor (TIMP‐4) causes diabetic cardiomyopathy is regulated by dicer, 10 weeks old male Ins2‐/+ Akita and C57 BL/6J mice were used to assess gravimetric parameters, physiological status of heart using pressure‐volume (P‐V) loop and echocardiography. Expression of GLUT‐4, MMP‐9, TIMP‐4 and dicer‐ a microRNA maturation enzyme was measured by RT‐PCR and Western Blot. Further, Akita mice were treated with folic acid (0.03 g/l) for 4 wks. The results revealed that Akita mice were hyperglycemic (>300 mg/dl) with low body weight/heart weight ratio as compared to control. Dilated cardiomyopathy of Akita mice was evident through echocardiography where systolic diameter was increased and fractional shortening was decreased. Additionally, impairment of ventricular contractility in Akita mice was confirmed by shift of P‐V loop towards right hand side. There was robust increase in MMP‐9 and dicer expression in Akita mice while TIMP‐4 and GLUT‐4 were decreased. Interestingly, folic acid treated Akita mice showed amelioration of MMP‐9 and TIMP‐4 expression. Based on these findings, we conclude that balance between MMP‐9 and TIMP‐4 is crucial for diabetic cardiomyopathy where dicer plays a regulatory role, and folic acid have therapeutic implications.
Store‐operated Ca2+ influx predicts coronary artery disease and is induced by dyslipidemia in metabolic syndrome and type 2 diabetes
Authors: Neeb Zachary P , Alloosh Mouhamad , Edwards Jason , Bratz Ian N. , Sturek Michael
Published: June 2021

Objective

We tested the hypothesis that dyslipidemia was the principal component of metabolic syndrome (MetS; “prediabetes”) in the progression to type 2 diabetes and increased store‐operated Ca2+ entry (SOCE) in coronary smooth muscle that contributes to coronary artery disease (CAD). Accordingly, we focused on and eliminated dyslipidemia from MetS allowing the contribution of dyslipidemia to be determined.

Methods and results

Ossabaw swine, an excellent humanoid model of MetS, were fed normal chow (Lean; N=9), high fructose diet that induced normolipidemic MetS (NMetS; N=6), or high fructose/trans‐fat/cholesterol diet that induced dyslipidemic MetS (DMetS; N=7) for 24 weeks before being assessed for MetS and CAD. NMetS did not develop MetS, type 2 diabetes, increased SOCE, or CAD. DMetS developed MetS with type 2 diabetes, increased SOCE, and CAD. Swine fed the same atherogenic diet as DMetS, but short‐term (sDMetS; N=3), developed dyslipidemic MetS with increased SOCE preceding CAD. Electrophysiology of coronary smooth muscle revealed absent Ca2+‐induced cation conductance and increased monovalent cation (Na+) permeance via store‐operated channels in DMetS compared to Lean.

Conclusions

Taken together, our results demonstrate dyslipidemia is the primary contributor to increased SOCE and the development of type 2 diabetes that precedes CAD in the metabolic syndrome.
Epicardial perivascular adipose tissue exacerbates coronary endothelial dysfunction in metabolic syndrome via leptin‐induced activation of PKC‐β
Authors: Payne Gregory Allen , Borbouse Lena , Kumar Sajel , Neeb Zachary , Alloosh Mouhamad , Sturek Michael , Tune Johnathan D.
Published: June 2021

This study tested the hypothesis that augmented release of leptin from epicardial perivascular adipose tissue (PVAT) exacerbates coronary endothelial dysfunction in metabolic syndrome (MetS) via a PKC‐β pathway. Coronary arteries with and without PVAT were collected from lean or MetS Ossabaw swine. Isometric tension studies demonstrated endothelial‐dependent vasodilation to bradykinin was reduced by MetS alone. PVAT did not affect vascular function in lean arteries, but exacerbated endothelial dysfunction by 28% in MetS arteries. This effect of PVAT was significantly attenuated by inhibition of either PKC‐β with ruboxistaurin (1 μM) or leptin receptor (ObRb) signaling with a recombinant, pegylated leptin antagonist (1 μM). Western analyses revealed a 50% increase in leptin expression in MetS PVAT, and a 20% increase in MetS coronary ObRb density. Immunohistochemistry confirmed ObRb expression in coronary endothelium. Leptin alone (30 ng/ml) diminished endothelial‐dependent dilation in lean arteries without PVAT and increased PKC activity > 3‐fold. PKC activity was also increased ~ 3‐fold in coronary arteries from MetS vs. lean swine. These effects were reversed by pre‐treatment with ruboxistaurin (1 μM). In conclusion, increased epicardial PVAT leptin expression exacerbates coronary endothelial dysfunction in MetS primarily via a PKC‐β‐dependent pathway. (Support: HL092245; HL62552; RR13233)
Impaired mesenteric resistance artery relaxation to KATP channel activation in cardiometabolic syndrome is improved by rosiglitazone treatment
Authors: Walsh Katie Ann , Carpenter Margaret A , Imig John D
Published: June 2021

Cardiometabolic syndrome is characterized by a combination of medical disorders that increase the risk for cardiovascular diseases. Here, we investigate the effect of the KATP opener, pinacidil and the KCa channel opener, NS1619 on mesenteric resistance arteries from spontaneously hypertensive obese rats (SHROB). Additionally, we assessed the ability of the soluble epoxide hydrolase inhibitor, tAUCB or the PPAR‐γ agonist, rosiglitazone (RGZ) to improve K+ channel vasodilation. SHROB rats were treated with RGZ or tAUCB [10mg/kg/d] for 4 weeks. Blood pressure was increased in SHROB (154±3 mmHg) and was not decreased by tAUCB or RGZ. Mesenteric arteries were isolated and diameter responses to K+ channel openers determined. Mesenteric artery diameters and dilator responses to NS1619 did not differ between WKY, SHR and SHROB. KATP channel dilator responses to 30uM pinacidil were attenuated in SHROB (76±6%), compared to WKY (105±2%) and SHR (92±12%). Vasodilation to pinacidil was greatly improved by RGZ (92±7%) but not tAUCB. Interestingly, Kir6.1 (KATP subunit) protein expression in mesenteric arteries was increased 5 fold in SHROB. These findings indicate that vasodilation to the KATP opener, pinacidil, is impaired despite increased Kir6.1 expression in SHROB. Furthermore, mesenteric artery KATP vasodilation was improved with RGZ, indicating a vascular benefit in cardiometabolic syndrome.
NFAT5 positively regulates SMαA gene expression in vascular smooth muscle
Authors: Lemmon Julia Ann , Kwon H Moo , Wamhoff Brian R
Published: June 2021

Nuclear factor of activated T‐cells 5 (NFAT5) is a tonicity‐responsive transcription factor; however, NFAT5 function in vascular SMCs is unknown. The SM α‐actin (SMαA) gene contains an NFAT5 cis element overlapping the first intronic CArG. We hypothesized that NFAT5 positively regulates SMαA gene expression through binding to this consensus sequence. NFAT5 overexpression increased SMαA promoter‐reporter activity, which was attenuated by mutating the NFAT5 site. siRNA knockdown of endogenous NFAT5 inhibited an Ang II‐mediated upregulation of SMαA promoter activity. Conversely, platelet‐derived growth factor‐BB decreased SMαA expression concomitant with a 70% decrease in NFAT5 binding to the SMαA first intron. In the A404 SM precursor cell line, treatment with retinoic acid to induce SMαA mRNA was preceded by increased NFAT5 expression. Microarray analysis of NFAT5 null mouse embryonic fibroblasts indicated a 64% decrease in SMαA mRNA compared to WT. Additionally, aortas from heterozygous NFAT5 mice have a 41% decrease in SMαA protein at 5 wks of age, returning to WT levels by 20 wks. In the rat carotid artery balloon injury model, NFAT5 protein was upregulated at 7 and 21 days, corresponding with an increase in SMαA protein back to basal levels and resolution to the contractile phenotype. In conclusion, we have identified NFAT5 as a novel positive regulator of SMαA gene expression in vascular SMCs. (NIH HL81682)
Interference with Peroxisome Proliferator Activated Receptor Gamma (PPARG) in smooth muscle causes aortic dysfunction via a Rho‐kinase‐dependent mechanism
Authors: Pelham Christopher James , Ketsawatsomkron Pimonrat , Groh Severine , de Lange Willem J. , Faraci Frank M. , Sigmund Curt D.
Published: June 2021

Transgenic mice expressing a vascular muscle‐specific dominant negative mutant of PPARG (S‐P467L) develop hypertension and aortic dysfunction, supporting a protective role for PPARG in the vasculature. Aortic rings from S‐P467L mice displayed impaired vasodilation to acetylcholine (Ach) and sodium nitroprusside (SNP); and augmented contraction to endothelin‐1 (10nM ET‐1; 0.54±0.04g vs 0.16±0.03g non‐transgenic, NT. P<0.01) that was blocked by pre‐incubation with a Rho‐kinase inhibitor (1μM Y‐27632 + 10nM ET‐1; 0.13±0.02g vs 0.03±0.02g NT). Maximal contraction to PGF2α or KCl was not altered. We tested the role of basal nitric oxide (NO) as an endogenousinhibitor of RhoA/Rho‐kinase signaling. The level of total and p‐Ser1177 eNOS protein did not differ between S‐P467L and NT aorta. NOS inhibition (L‐NAME 100μM) increased contraction of S‐P467L aorta to ET‐1 (1.30±0.06g) to a greater degree than NT (0.50±0.05g, P<0.01). Similar results were observed for PGF2α and KCl. Rho‐kinase‐dependent phosphorylation of MYPT was enhanced in S‐P467L aorta (172±10% of NT, P<0.01). Pre‐incubation with Rho‐kinase inhibitor (1μM Y‐27632) significantly improved relaxation responses to Ach and SNP in S‐P467L aorta, but did not affect NT. We conclude that interference with PPARG in vascular muscle promotes aortic constriction through elevated Rho‐kinase activity, which is inhibited by endogenous NO.
Proliferation of Macula Densa cells in Response to Succinate
Authors: Blebu Bridgette E , Toma Ildiko , Vargas Sarah L , Koo Jahoo J , Peti‐Peterdi Janos
Published: June 2021

The kidney normally functions under the stress of hypoxia, but when coupled with diabetes mellitus, the altered metabolism in the citric acid cycle leads to robust accumulation of intra‐renal succinate. Succinate is the ligand for the newly identified metabolic receptor GPR91 that signals through MAPK Erk1/2 and p38 in macula densa cells (MD), a special cell type in the kidney which controls renal blood flow, glomerular filtration and the renin‐angiotensin system. The MAPK system is well‐established to trigger cell proliferation and also involved in diabetic nephropathy. Here we aimed to study whether succinate via GPR91 causes MD cell proliferation and altered MD signaling. Mouse macula densa‐derived cells (MMDD1) were grown to 70% confluency and treated with succinate (10 uM, 100 uM and 1 mM) for 24‐hrs, followed by treatment with a fluorogenic proliferation assay reagent. Succinate caused a dose‐dependent proliferation of MMDD1 cells with a peak 2.6‐fold increase in cell number at 1 mM. There was increased MD COX‐2 expression in the STZ‐diabetic kidney which was GPR91‐dependent. Our new findings suggest a link between MD GPR91 expression and cell proliferation, MAPK and COX‐2 signaling in diabetes.
Modulation of PI3‐K/AKT pathway by renin‐angiotensin system inhibition in obesity experimental model.
Authors: Carvalho Diego Soares , Alves Eduardo Silva , Haidar Andre Abou , Quadros Caren D′Mora , Morgan Daniela , Rocha Marlene Santos , Carpinelli Angelo Rafael , Hirata Aparecida Emiko
Published: June 2021

Insulin resistance (IR) is considered the main pathophysiological feature of metabolic syndrome that is defined by a set of abnormalities including glucose intolerance, hypertension, abdominal obesity and dyslipidemia. Several studies have shown that angiotensin II receptor blockers (ARBs) such as losartan, improve IR and that the adipose RAS may play important role in the pathogenesis of obesity, obesity‐associated hypertension. To determine this, we studied PI3‐K/AKT activation on MSG‐obese rats after 8‐day‐losartan treatment by gavage. Western blotting analysis showed that losartan treatment increased AKT phosphorylation on muscle of MSG‐obese rats which mechanism may be responsible for the improvement in insulin resistance. However adipose tissue of the obese animals presented significant decrease in IRS2/PI3‐K interaction and AKT phosphorylation. ARBs are now widely used as therapy for hypertension in diabetic patients and appear to involve PPARs activation. We can suggest that ARBs treatment improve muscle insulin resistance and lipid metabolism and those such effects may be via PPARs activation.
Exacerbation of endothelial dysfunction in aged diabetic mice
Authors: Yang Yang‐Ming , Huang An , Wu Hongyan , Bagi Zsolt , Kaley Gabor , Sun Dong
Published: June 2021

To test the hypothesis that aging accelerates endothelial dysfunction in type 2 diabetes mellitus, shear stress (SS)‐induced dilation (1, 10 and 20 dyne/cm2) and release of nitric oxide (NO) were determined in mesenteric arteries (~200–250 μm in diameter) isolated from three‐ (3M) and nine‐month (9M) old male db/db mice and their wild type (WT) controls. NO‐mediated SS‐induced dilation was significantly attenuated in arteries of 3M‐db/db mice and reduced further in those of 9M‐db/db mice compared to their age‐matched controls, in which, the dilation was comparable. SS‐stimulated release of nitrite was also significantly reduced in db/db mice compared to WT mice. Moreover, the magnitude of the reduction was greater in 9M compared to 3M‐db/db mice. Western blot analysis indicated significantly decreased SS‐induced phosphorylation of Akt (p‐Akt), as well as p‐eNOS, accounting for the reduced SS‐stimulated NO release and dilation in db/db mice compared to WT mice, and in 9M‐ compared to 3M‐db/db mice. Vascular superoxide production was significantly increased in 3M‐db/db and even more in 9M‐db/db mice. The levels of enhanced superoxide in both groups of db/db mice were proportional to increases in NOX‐2 protein expression. In conclusion, decreased NO bioavailability is responsible for the endothelial dysfunction in arteries of db/db mice that becomes further aggravated with aging (supported by NIH HL43023 and HL 070653).
Indomethacin enhances reactivity to vasopressin in normotensive and hypertensive rats, while PGE2 receptor blockade only attenuates reactivity in normotensive rats.
Authors: Sellers Minga M , Stallone John N
Published: June 2021

Previously, we reported that in female (F) rats 17‐β‐estradiol and aortic coarctation‐induced hypertension (ACIH) increase release of thromboxane (TXA2) and prostacyclin from mesenteric arterioles (MA), and that constrictor responses of aorta to vasopressin (VP) are reduced ~40% by TP receptor antagonist SQ 29,548 (SQ). Thus, the roles of TXA2 and PGE2 in constrictor responses to VP were studied in MA. F Sprague‐Dawley rats (14–16 wks age) underwent ACIH (hypertensive; HT) or sham (normotensive; NT). Rats were sacrificed 12–14 days post‐ACIH and reactivity to VP was measured in MA (150–200 μm dia.) using isometric myography. Data are means Â± S.E. (n = 4–5 rats/group). Maximal contractile responses of MA to VP did not differ between NT (9.97 ± 0.75 mN/mm) and HT (11.77 ± 1.40). SQ (40 μM) did not alter responses in NT (9.16 ± 1.53) or HT (12.69 ± 1.77). In separate rats, maximal responses to VP did not differ between NT and HT. AH6809, a PGE2 receptor blocker (EP1, 2, 3) reduced maximal responses of MA from NT (8.95 ± 0.36 vs. 10.24 ± 0.63) but not HT (9.73 ± 0.81 vs. 9.36 ± 0.61). Indomethacin (non‐specific COX inhibitor; 10 μM) increased contractions to VP in NT and HT (11.74 ± 0.84 and 12.75 ± 0.79, respectively). While TXA2 potentiates constrictor responses of aorta to VP, the role of TXA2 in reactivity of MA to VP is less certain. However, it appears that PGE2 may also play a role in modulating MA responses to VP in NT rats. (NIH:HL‐080402)
Role of NO in Hyperuricemia induced Cardiac Metabolic Dysfunction in Pregnancy.
Authors: Martin‐Fernandez Beatriz , Song Su , Huang Har‐Er , Hintze Thomas
Published: June 2021

Hyperuricemia (HU) causes cardiovascular dysfunction and is associated with many complications during pregnancy related to reduced NO bioactivity and high blood pressure (BP). This study assessed the mechanisms of HU on the NO‐dependent control of cardiac O2 consumption in pregnant rats. Pregnant rats were fed oxonic acid (OA), an uricase inhibitor, (750mg/Kg body weight/day) by gavage (OA group) and allopurinol (AL), a xanthine oxidase inhibitor (OAA group), was placed in drinking water (150mg/L) every day for 20 days. OA increased significantly mean blood pressure (89.91±7.8 to 110.38±5.6 mmHg), heart weight/body weight ratio (0.24±0.01 to 0.26±0.01g/100g) and total peripheral resistance (1.15±0.15 to 1.55±0.17 mmHgmin−1m−1). Treatment with AL reduced this response. The ability of bradykinin and carbachol to reduce myocardial O2 consumption in vitro was impaired by 70% and 50%. The response was restored by tiron, tempol and apocynin. Pregnant rats fed L‐NAME also have impaired ability of bradykinin and carbachol to regulate myocardial O2 consumption (59% and 62%). In summary, HU induces hypertension, increases O2 production and reduces the ability of NO to regulate myocardial O2 consumption in pregnancy. Supported by HL43023, 50142.
Lung arginase activity is increased in ovine model of acute lung injury and contributes to long term pulmonary dysfunction
Authors: Sousse Linda , Yamamoto Yusuke , Enkhbaatar Perenlei , Yu Young‐Ming , Rehberg Sebastian , Traber Lillian , Herndon David , Traber Daniel
Published: June 2021

Thermally injured children who have sustained inhalation injury show evidence of restrictive lung disease during their recovery period. We hypothesize that lung arginase activity is increased by burn and inhalation injury, leading to increased collagen deposition in the lung and contributing to long‐term pulmonary dysfunction.

METHODS

Ewes were randomly divided into (1) Uninjured: n=6 and (2) injured: n=7 groups. The injured animals were subjected to 20% TBSA burn and 36 breaths of cotton smoke under deep anesthesia.

RESULTS

DDAH‐II, an enzyme that degrades ADMA, which is an endogenous inhibitor of NOS, significantly decreases after 2 and 3 weeks in the lung compared with uninjured animals. Lung ADMA increases from 2 to 3 weeks. Expression of iNOS significantly decreases in the lung 2 weeks after injury compared with uninjured animals. Lung arginase activity and hydroxyproline, a marker of collagen deposition, increase with 3 weeks post‐injury compared to the uninjured animals. The increases in arginase and collagen are associated with reduced gas exchange and lung diffusion capacity after 3 weeks compared to uninjured animals.

CONCLUSION

The decrease in DDAH‐II results in increased ADMA, resulting in decreased NOS. The decrease NOS causes an increase in arginase activity because NOS forms NOHA, which forms NO and inhibits arginase. The increase in arginase activity contributes to the increase in collagen deposition, which leads to long‐term pulmonary dysfunction. Interruption of this pathological chain reaction may be useful in preventing the long‐term sequelae of the smoke inhalation and burn injury.
Carbon monoxide inhibits tubuloglomerular feedback in vivo by activation of soluble guanylyl cyclase
Authors: Wang Hong , Garvin Jeffrey L. , Carretero Oscar A.
Published: June 2021

We have previously shown in vitro that carbon monoxide (CO) inhibits tubuloglomerular feedback (TGF), but how this happens is not known. We hypothesized that CO in vivo inhibits TGF by activating the soluble guanylyl cyclase (sGC)/cyclic guanosine 3′, 5′‐monophosphate (cGMP) system. Renal micropuncture studies were performed in male Sprague Dawley rats. TGF was induced by increasing late proximal perfusion rate (0, 10, 20, 30, 40 nL/min) while measuring stop‐flow pressure (PSF) as an index of single nephron glomerular filtration. We measured the effect of a CO‐donor, CORM‐3 (50 μM) on PSFmax with and without inhibition of sGC using the sGC inhibitor LY83583 (1 μM). First, we demonstrated that the PSF response to increased tubular perfusion rate was reversible and reproducible. Next, we found CORM‐3 blunted the TGF response, as the maximum change in PSF was 6.9 ± 0.2 mmHg in controls, but only 3.9 ± 0.4 mmHg in CORM‐3 treated tubules (p < 0.001). However, with addition of LY83583 to the perfusate, the effect of CORM‐3 was eliminated. Addition of LY83583 potentiated the TGF response by 28% (8.4 ± 0.3 mmHg, p < 0.001), and it also reversed the blunting effect of CORM‐3 (8.3 ± 0.4 mmHg, p < 0.001) to a similar extent. From these data, we conclude that inhibition of TGF by CO in vivo is mediated by activation of the sGC/cGMP system. This study was supported by National Institutes of Health Grant HL‐090550.
Interstitial adenosine but not nitric oxide concentrations are elevated during systemic hypoxia in rat skeletal muscle
Authors: Smith Ashley A. , Zhao Yi , Fabris Sergio , MacLean David A.
Published: June 2021

During systemic hypoxia it is generally accepted that nitric oxide (NO) and adenosine (Ado) contribute to peripheral vasodilatation in skeletal muscle. To examine this, microdialysis fibers were inserted into the gastrocnemius muscle of anesthetized rats (n = 16) and perfused with ringers (control), 60 μM ATP (Ado precursor) or 500 μM AOPCP (5′ectonucleotidase inhibitor) at a rate of 5 μL/min during rest and hypoxia (10.5% O2). During all perfusions and conditions no changes in interstitial NO concentrations were observed. Interstitial Ado levels during ringers perfusion were 0.04±0.01 μM at rest and increased to 0.07±0.01 μM during hypoxia (P<0.05). The perfusion of ATP alone increased (P<0.05) interstitial Ado levels from 0.04±0.01 μM to 0.12±0.02 μM at rest and hypoxia further increased (P<0.05) Ado levels to 0.18±0.02 μM. Perfusion of AOPCP reduced interstitial Ado to undetectable levels throughout the experiment, confirming inhibition of the 5′ectonucleotidase and subsequently Ado production. These data clearly demonstrate that hypoxia and alterations in Ado concentrations do not directly affect NO concentrations. In particular, when Ado production was inhibited, NO did not compensate for the loss of this vasodilator, even during hypoxia. Furthermore, interstitial Ado levels were elevated during hypoxia and appear to play a key role in interstitially mediated vasodilation.

Supported by NSERC.
The contribution of guanylyl cyclase and potassium channels to the relaxation induced by the NO donors [Ru(terpy)(bdq)NO+]3+ (Terpy) and sodium nitroprusside (SNP) in rat mesenteric resistance arteries
Authors: Araújo Alice Valença , Vercesi Juliana Aparecida , Biazzotto Juliana Cristina , da Silva Roberto Santana , Bendack Lusiane Maria
Published: June 2021

This study aimed to verify the mechanisms of vasodilatation induced by Terpy and SNP in resistance mesenteric arteries isolated from normotensive (2K) and renal hypertensive rats (2K‐1C). We also studied the inhibitory effects of the guanylyl‐cyclase (GC) with ODQ and K+ channel with TEA. Cumulative concentration‐effect curves to Terpy (0.01 μM‐ 0.01mM) and sodium nitroprusside (SNP 1nM‐0.1mM) were constructed in the third branch of mesenteric arteries pre‐contracted with phenylephrine. We have analyzed the efficacy (ME) and the potency (pD2) of the NO donors. Both SNP and Terpy induced relaxation with similar efficacy in 2K (Terpy 93.8±1.2%, n=7 SNP 96.1±0.7% n=6) and 2K‐1C rats (Terpy 93.7±1.4% n=8, SNP 90.2±4.2% n=5). However, Terpy was less potent (pD2) (2K 4.45±0.3; 2K‐1C 4.51±0.3) than SNP (2K: 6.60±0.2, 2K‐1C: 7.28±0.01), although the relaxation induced by Terpy and SNP, were not different in mesenteric resistance arteries from 2K and 2K‐1C. The incubation with ODQ (1μM) almost abolished the response induced by Terpy and SNP. TEA (1mM) reduced the effect of Terpy in a similar way in 2K (ME: Terpy 73.1±7.3% n=5; SNP 69.2±6.2% n=7) and 2K‐1C rats (Terpy 71.7±8.2% n=5; SNP 77.1±6.6% n=7). In conclusion, in mesenteric resistance arteries from hypertensive and normotensive rats the NO donors Terpy and SNP induce relaxation in a similar way, which involves GC and K+ channels activation. Supported by FAPESP and CNPq.
Vasoregulation by isoform‐selective coupling of TRPC3 channels to IP3 receptors in smooth muscle cells
Authors: Adebiyi Adebowale , Zhao Guiling , Narayanan Damodaran , Thomas Candice M , Bannister John P , Jaggar Jonathan H
Published: June 2021

Inositol 1,4,5‐trisphophate (IP3) stimulates vasoconstriction independently of intracellular Ca2+ release via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but signaling mechanisms mediating this effect are unclear. Here, we studied the mechanisms by which IP3Rs stimulate TRPC channels in cerebral artery myocytes. Immuno‐FRET microscopy in myocytes indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels. Endothelin‐1 (ET‐1), a PLC‐coupled receptor agonist, elevated the immuno‐FRET signal between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. IP3R1 co‐immunoprecipitated with TRPC3, but not with TRPC6. An antibody targeting TRPC3 channels and TRPC3 channel knockdown using shRNA inhibited IP3‐induced non‐selective cation current (ICat) activation, whereas an antibody to TRPC6 and TRPC6 channel knockdown had no effect. Biotinylation indicated that ET‐1 did not alter total or plasma membrane‐localized TRPC3. RT‐PCR demonstrated that a calmodulin and IP3R binding (CIRB) domain is present on the C‐terminus of both TRPC3 and TRPC6 channels. A peptide corresponding to the IP3R region that can interact with TRPC channels activated ICat. A CIRB domain peptide attenuated IP3‐ and ET‐1‐induced ICat activation and vasoconstriction. These data indicate that IP3 stimulates direct coupling between IP3R1 and membrane‐resident TRPC3 channels in arterial myocytes, leading to ICat activation and vasoconstriction. Data also indicate that close spatial proximity between IP3R1 and TRPC3 establishes this isoform‐selective functional interaction.
Organ Culture: Effect on Fetal and Adult Cerebral Arterial Contractility
Authors: Goyal Ravi , Chu Nina , Goyal Dipali , Longo Lawrence D.
Published: June 2021

Small interfering RNA (siRNA) can be used to knockdown protein expression of several genes. However, in vascular smooth muscle, to produce siRNA‐mediated silencing of protein expression, arteries need to be maintained in serum‐free media (organ culture). Moreover, organ culture leads to significant changes in phenotype, which may alter contractility. Thus, we tested the hypothesis that serum‐free organ culture leads to changes in phenotypes and agonist‐induced contractile responses in fetal and adult cerebral arteries. To study effects on phenotypic changes from contractile to synthetic phenotype, we maintained cerebral arteries for 72 hrs in serum‐free media. We examined phenotype markers protein and mRNA expression, as well as contractile responses to KCl, serotonin (5‐HT) and phorbol 12,13‐dibutyrate (PDBu). Phenotype changes, and KCl, PHE and PDBu ‐mediated contractile responses are altered during 24 to 48 hours of serum‐free organ culture. However, by 72 hours the ratio of contractile and synthetic phenotype markers as well as contractile responses to different agonists, reaches similar to that of 0 hours. Thus, we demonstrate that serum free organ culture can be used for in‐vitro studies by 72 hours. Supported by NIH Grant HD3807‐37 to LDL.
Smad3 inhibits myocardin expression in the initial stage of smooth muscle differentiation
Authors: Chen Shi‐You , Xie Weibing , Long Xiaochun , Miano Joseph M
Published: June 2021

The objective of this study is to determine the precise role of Smad and myocardin in the early stage of TGF‐β‐induced smooth muscle (SM) differentiation. By using TGF‐β‐induced pluripotent C3H10T1/2 cells as a model, we found that TGF‐β did not induce myocardin expression until 18 hrs of stimulation. SM22α promoter activity, however, was activated within 4 hr, and reached the highest level within 8 hours of TGF‐β induction. Endogenous SM22α was detectable 2 hours and reached the highest level 8 hours after TGF‐β treatment. These data suggest that myocardin is not involved in the initiation of TGF‐β‐induced SM differentiation. In consistent with the endogenous mRNA expression, myocardin promoter could not be activated until 18 hrs after TGF‐β induction. Interestingly, Smad3 overexpression inhibited myocardin promoter activity. To test if endogenous Smad3 blocks myocardin promoter in the early stage of SM differentiation, dominant negative Smad4 ( Smad4) was used to prevent Smad3 function. Blockade of Smad3 allowed TGF‐β to activate myocardin promoter in the first few hours of induction. These data indicate that Smad3 inhibits myocardin expression in the onset of SM differentiation program, which is likely an essential mechanism governing the precise regulation of the initiation of phenotypic conversion. (Funding source: NIH R01HL093429)
Second Messenger Communication and the Regulation of Vascular Contractility
Authors: Tran Cam Ha , Plane Frances , Taylor Mark , Welsh Donald
Published: June 2021

This study examined the role of second messenger communication in the regulation of vascular contractility. We specifically hypothesized that the generation of inositol 1,4,5 trisphosphate (IP3) by smooth muscle‐dependent vasoconstrictors sequentially cross myoendoendothelial gap junctions (MEGJs), elicits Ca2+ release from IP3 receptors (IP3R) and activates intermediate conductance Ca2+ sensitive K+ channels (KCa3.1) to elicit a negative feedback response. Work began by monitoring the vasomotor/electrical responses of hamster retractor muscle feed arteries to an increasing concentration of superfused phenylephrine (PE) in the absence/presence of KCa2.3, KCa3.1 or an IP3R inhibitor. Consistent with the stated hypothesis, KCa3.1 and IP3R blockade increased the contractile and electrical responsiveness of feed arteries to superfused PE, whereas KCa2.3 inhibition had no significant effect. These changes were absent in vessels constricted to 4‐AP, a K+ channel blocker that elicits a receptor independent depolarization. Electron microscopy along with immunohistochemistry confirmed the presence of MEGJs and the punctated expression of KCa3.1 channels and IP3R1/IP3R2 in endothelial cells. Further, endothelial Ca2+ imaging on intact and opened vessels confirmed the presence of IP3R‐driven Ca2+ waves that displayed some sensitivity to global PE application. In summary, we conclude that IP3 signalling between smooth muscle and endothelial cells contributes to a negative feedback response that moderates agonist‐induced constriction. Supported by HSFC and AHFMR.
Endothelin‐1 induces smooth muscle cell plasticity in small mesenteric arteries
Authors: Schoorl Marieke , Bakker Erik N , de Vos Judith , vanBavel Ed
Published: June 2021

Aim

Small arteries show eutrophic inward remodeling in response to many physiological and pathological stimuli. We hypothesized that early events include repositioning and adapted architecture of smooth muscle cells, reflected by shifts in active length‐tension relations.

Methods

Rat mesenteric arteries were incubated overnight in a wire myograph at low (0.4 D100) or high (1.1 D100) distension with or without 10 nM endothelin‐1 (ET‐1). D100 is the passive diameter at an equivalent pressure of 100 mmHg. Before and after incubation, active (125 mM K+) and passive force‐length relationships were determined by stepwise distension of the vessel.

Results

Segments (n=10) incubated with ET‐1 at 0.4 D100 showed a marked leftward shift of the force‐length curve. Optimal force developed at 0.90±0.02 D100 (day 0) versus 0.84±0.02 D100 (day 1, p=0.03). This shift did not occur in the absence of ET‐1. At 1.1 D100 incubation, slight rightward shifts occurred.

Conclusion

maintained activation at low diameter shifts tension‐generating properties, possibly forming an early event in inward remodeling. This study was performed within the framework of the Dutch Top Institute Pharma project T2‐108.
Molecular mechanisms of Serotonergic Potentiation of Endothelin‐1: involvement of the JAK2 and p42/44 MAPK pathways.
Authors: Telraja Deepa , Bhaskaran Subha , Ahmad Zeinab , Day Jennifer , Banes‐Berceli Amy K.L.
Published: June 2021

We investigated the molecular mechanisms of serotonin's (5‐HT) ability to potentiate endothelin‐1 (ET‐1) stimulated cellular proliferation and contraction. Previous data suggests that 5‐HT/ET‐1 potentiation mechanism involves activation of the mitogen activated protein kinase (p42/44 MAPK) pathway. No data exists looking at the involvement of the Janus Kinase (JAK) pathway in this mechanism. We tested the hypothesis that both the JAK and p42/44 MAPK pathways were involved in the potentiation. Using rat aortic vascular smooth muscle cells (RAVSMC) we pre‐incubated with 5‐HT and then added ET‐1 for 10 min. Our Western blot data demonstrates that 5‐HT and ET‐1 at a concentration of 10 nM can elicit this potentiation in RAVSMC. There were significant increases in the levels of phosphorylation for both the JAK2 and p42/44 MAPK when combined and no effect when incubated separately. Additionally, in isolated vessel studies (thoracic rat aorta and superior mesenteric arteries) in a myograph we measured the isometric tension generation. We found that 1 nM 5‐HT pre‐incubation leftward shifted the EC50 value of the ET‐1 concentration response curve and significantly increased the maximal response. These changes were inhibited by the JAK2 inhibitor AG490 and the p42/44 MAPK kinase inhibitor UO126. These data suggest that both the JAK2 and p42/22 MAPK pathways are involved in these potentiation mechanisms. (ABB NHLBI R00).
Intracellular calcium stores contribute to the myogenic response of rat cerebral arteries
Authors: Tam Raymond , Kerr Paul M , Plane Frances
Published: June 2021

Cerebral blood flow is controlled by autoregulatory mechanisms which maintain blood flow within narrow limits despite large changes in blood pressure. Increases in blood pressure cause small cerebral arteries to constrict, a mechanism called the myogenic response. We investigated the contribution of intracellular calcium stores to the development of myogenic tone in rat middle cerebral arteries.

Cerebral arteries mounted in a pressure myograph were subjected to stepwise increases in intra‐vascular pressure (20–120 mmHg), and changes in vessel diameter were recorded in the presence or absence of drugs. Cyclopiazonic (CPA; 10 μM), an inhibitor of the SR Ca2+‐ATPase, attenuated myogenic tone at 80mmHg by 64.2 ± 7.2% (n=5). Caffeine (10 mM), a ryanodine channel opener, reduced myogenic tone by 80.3 ± 6.7% (n=5). The effect of caffeine was not inhibited by iberiotoxin (IBTX 100nM; n=4), a large conductance calcium‐activated potassium channel (BKCa) blocker, suggesting that activation of BKCa is unlikely. When CPA was added in combination with nifedipine (10 μM), an L‐type calcium channel blocker, development of myogenic tone was abolished. In conclusion, our data suggests that release of intracellular calcium from the SR plays an important role in the development of myogenic tone in rat middle cerebral arteries. Funded by Heart and Stroke Foundation and University of Alberta Queen Elizabeth II Award.
Modulation of ATP release in gastrocnemius muscle 1A arterioles
Authors: Kluess Heidi A , Stone Audrey J , Evanson Kirk W
Published: June 2021

The purpose of this study was to determine the possible sources of ATP in the synapse of 1A arterioles and to evaluate the role of α2‐pre‐junctional receptors in ATP modulation. Using F344 rats (6 months old), the gastrocnemius 1A arterioles from the red gastrocnemius muscle were dissected and cannulated with glass pipettes in a tissue bath. The ATP and null probes were positioned such that the tips of the probes were touching the arteriole in similar anatomical locations and a normal ATP curve was generated (0.78μM to 25 μM ATP). The arterioles (n=3) were then field stimulated at 60Hz (200 impulses, 9mA) which resulted in ATP production of 0.54±0.76uM. This was abolished by tetrodotoxin (10uM). In a separate group of rats (n=6), the first 20 sec following 60Hz stimulation resulted in 5.1±6uM ATP production, the last 30 seconds resulted in 6.5±15.5uM ATP production. Adding the α2‐agonist, UK14,304, resulted in 7.7±7.8uM ATP in the first 20 sec and 14.7±26.0uM ATP in the last 30 seconds. Blocking α2‐receptors using idozoxan, resulted in an increase in ATP in the first 20 seconds (12.1±20.3uM ATP), but no change in the last 30 seconds (15.9±14.4uM ATP). These data suggest that the primary source of ATP in skeletal muscle arterioles is from the sympathetic nerve. Activation of pre‐junctional α2‐receptors increases late ATP release, but blocking α2‐receptors appears to increase early ATP release. This research was supported by the Arkansas Biosciences Institute and the National Institute on Aging.
Regulation of voltage‐gated calcium channel expression by estrogen
Authors: Dalton Jamie , Joseph Biny K. , Rusch Nancy J. , Hill Brent J.F.
Published: June 2021

We previously demonstrated that a physiological estrogen (E2) concentration decreases the protein expression of the voltage‐gated calcium channel (VGCC) pore‐forming α1C subunit in coronary arteries. However, it is not known how E2 elicits this effect. In this study we hypothesized that the E2 mediated down‐regulation occurs via a posttranscriptional pathway in arterial smooth muscle. Right coronary arteries were obtained from hearts of mature sows. The distal end of the artery was cut into longitudinal strips, and the endothelium was left intact or mechanically removed. The strips were incubated for 24 hours in E2 (1 nM) or its ethanol solvent. Using RT‐PCR our data suggest that E2 has no effect on the mRNA expression of the VGCC α1C subunit. Preliminary results from Western blots indicate that the decrease in VGCC expression by E2 is endothelium‐independent. Our findings provide important insights into the mechanistic pathway of the E2 mediated down‐regulation of VGCCs. Because vascular disease is accompanied by endothelial damage, the presence of circulating E2 may have beneficial effects to limit coronary vasoconstriction.

Support: NCRR of the NIH, Grant #P20 RR‐16460
CaV1.2 N‐terminal splice variants regulate cerebral artery diameter
Authors: Bannister John Philip , Thomas Candice , Adebiyi Adebowale , Cheng Xiaoyang , Zhao Guiling , Jaggar Jonathan Harvey
Published: June 2021

Voltage‐dependent Ca2+ (Cav1.2) channels are the major Ca2+ entry pathway in arterial myocytes and are essential for myogenic constriction. Alternative splicing of CaV1.2 α1 subunit exon 1 generates two different N‐terminal variants in arterial myocytes (CaV1.2e1b and CaV1.2e1c). Here, we examined physiological functions of Cav1.2 α1 subunit N‐terminal splice variants in cerebral artery myocytes. Acute application of a membrane‐permeant peptide corresponding to the CaV1.2e1c N‐terminus inhibited myocyte voltage‐dependent Ba2+ currents and reversibly dilated pressurized arteries. Overexpression of the exon1‐encoded N‐terminus of CaV1.2e1b or CaV1.2e1c reduced pressure‐induced vasoconstriction (myogenic tone). shRNA targeting exon 1b selectively reduced CaV1.2e1b by ~39% and myogenic tone (at 60 mmHg) by ~34%. shRNA to exon 1c selectively reduced CaV1.2e1c by ~62% and myogenic tone by ~57%. shRNA targeting α2δ‐1, a CaV1.2 auxiliary subunit, reduced α2δ‐1 by ~57 % and plasma membrane‐localized (measured using biotinylation) CaV1.2e1b by ~54% and CaV1.2e1c by ~35%. Data indicate that both CaV1.2e1b and CaV1.2e1c modulate cerebral artery diameter and that α2δ‐1 differentially regulates plasma membrane trafficking of CaV1.2 N‐terminus variants. NIH HL67061, HL094378.
Effect of Lophatherum gracile Brongn on the mechanism of vasorelaxation in thoracic aorta
Authors: Kim Hye Yoom , Li Xiang , Kang Dae Gill , Lee Ho Sub
Published: June 2021

The vasorelaxant effect of an ethanol extract of Lophatherum gracile Brongn (ELB) and the possible mechanism were ascertained in aortic tissues isolated from rats. ELB relaxed endothelium‐intact thoracic aorta in a dose‐dependent manner. However, the induced vascular relaxation was abolished by removal in endothelium of the thoracic aorta. Pretreatment of endothelium‐intact vascular tissues with NG‐nitro‐L‐arginine methyl ester (L‐NAME) or 1H‐[1,2,4]‐oxadiazole‐[4,3‐¥á]‐quinoxalin‐1‐one (ODQ) significantly inhibited vascular relaxation induced by ELB. Moreover, ELB significantly increased cGMP production in aortic tissues, which was blocked by pretreatment with L‐NAME or ODQ. The vasorelaxant effect of ELB was attenuated by tetraethylammonium (TEA), and glibenclamide. ELB‐induced vasorelaxation was not blocked by atropine, propranolol, indomethacin, verapamil, and diltiazem. Taken together, the present study demonstrates that ELB dilates vascular smooth muscle via an endothelium‐dependent NO‐cGMP signaling pathway, which may be at least, in part, related with the function of K+ channels.
The Effect Of Ethanol On Tracheal Smooth Muscle Of Cat
Authors: Jakupaj Muharrem , Sopi Ramadan B , Lajçi Azem , Neziri Burim , Devaja‐Kumnova Shqipe , Krasniqi Avdyl
Published: June 2021

Previously we have shown that ethanol induces contraction of pulmonary artery smooth muscle (PASM). However, the effect of ethanol on tracheal smooth muscle (TSM) and the mechanisms involved are not well understood. Therefore, we hypothesized that ethanol induces contractile responses of TSM and extra cellular Ca2+ is involved to this effect. In vitro force measurements were performed in TSM strips, excised from cats (n=29). Tissues were stabilized in Krebs‐Henseleit (KH) solution and treated with different concentrations of ethanol (46 – 276 mM). The role of extra cellular Ca2+ was studied by incubation of tissue in a Ca2+ free KH solution or using a Ca2+ channel blocker‐nifedipine. The role of nitric oxide (NO) was assessed by inhibition of NO synthase (NOS) using Nω‐nitro‐L‐arginine methyl ester. The ethanol induced concentration‐dependent contraction of TSM. Lack of Ca2+ in medium resulted with decrease of TSM tone likewise Ca2+ channel blocker. Inhibition of NOS, significantly increased contractile responses of TSM to the ethanol at lower ad middle concentrations, but reduced these responses at higher concentrations. In addition, inhibition of NOS significantly potentiated TSM tone to ACh.

These data demonstrate that ethanol induces TSM contraction of cat and extra cellular Ca2+ is an important involved factor. NO release as a relaxant molecule counteracts the effect of ethanol and other agonists.
MYPT1 structure mediates PKGIα phosphorylation
Authors: Yuen Samantha , Ogut Ozgur , Brozovich Frank
Published: June 2021

During nitric oxide (NO) signaling, cGMP activates type Iα protein kinase G (PKGIα), which subsequently activates myosin light chain phosphatase (MLCP). The increase in MLCP activity decreases myosin phosphorylation which leads to smooth muscle relaxation. MLCP is a trimeric enzyme consisting of a myosin targeting subunit (MYPT1), a catalytic subunit and a subunit of unknown function. MYPT1 has two isoforms; alterative mRNA splicing of a 3’ exon results in the presence or absence of a C‐terminal leucine zipper (LZ) domain. Further, the MYPT1 LZ+ is required for PKGIα mediated activation of MLCP. Moreover, recent studies have demonstrated PKGIα phosphorylates MYPT1. This study examined the affect of MYPT1 structure for PKGIα phosphorylation. Two MYPT1 fragments differing only by the LZ domain (aa 1010–1039) were used: MYPT1FL (aa 500–1039, LZ+) and MYPT1TR2 (aa 500–1010, LZ−). Both the rate (0.0216±0.005 min−1 vs 0.0596±0.005 min−1) and extent of PKGIα mediated phosphorylation of MYPT1TR2 were lower when compared to MYPT1FL. In addition, two‐dimensional SDS‐PAGE demonstrated that following PKGIα phosphorylation, MYPT1TR2 existed primarily as the non‐phosphorylated protein whereas MYPT1FL was diphosphorylated. These results demonstrate that MYPT1 structure is important for determining the sensitivity of smooth muscle to NO mediated vasodilatation.
Increased PDK4 expression via PGC‐1α/ERRα – dependent mechanism in mouse skeletal muscle after high fat feeding
Authors: Rinnankoski‐Tuikka Rita , Torvinen Sira , Silvennoinen Mika , Lehti Maarit , Kivelä Riikka , Kainulainen Heikki
Published: June 2021

The key regulator of cellular energy metabolism is the peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α). It is shown that PGC‐1α coactivates the pyruvate dehydrogenase kinase (PDK4) gene expression via the estrogen related receptor (ERRα). Our aim was to investigate the effects of high fat (HF) feeding and physical activity in the expression levels of these genes in the skeletal muscles of C57BL/6J mice.

The HF animals showed an altered blood lipid profile suggesting changes in the overall lipid metabolism. The expression level of PGC‐1α was slightly down‐regulated after HF feeding, although voluntary wheel running restored the levels. Upregulation of ERRα and PDK4 was seen after HF diet and this combined with running had even more profound effects on the expression levels.

Although HF diet did not increase the PGC‐1α mRNA levels, we hypothesize that the regulation of energy metabolism after HF feeding leads to a situation where PDK4 inhibits the glucose oxidation coincident with the increased mitochondrial fatty acid oxidation. Whether the fatty acid oxidation is reduced de facto after HF feeding, as the hypothesis on accumulation of ectopic lipids might suggest, remains to be further clarified. The research was funded by the Finnish Ministry of Education.
Training induced improvements in aerobic capacity can occur independent of PGC‐1[alpha] in aging human skeletal muscle
Authors: Konopka Adam , Reidy Paul , Jemiolo Bozena , Kaminsky Leonard , Trappe Todd , Trappe Scott , Harber Matthew
Published: June 2021

The purpose of this investigation was to elucidate the molecular adaptations in skeletal muscle that occur with aerobic exercise training in elderly individuals. Muscle biopsies were obtained from the vastus lateralis of nine old women (70±2y) to determine basal mRNA and/or protein content of factors involved in mitochondrial biogenesis (TFAM, PGC‐1α, COX IV) before and after 12‐weeks of aerobic training on a cycle ergometer. Post training biopsies occurred ~48 hours after the last exercise session in order to assess steady state levels of select mRNA and/or protein. Whole body aerobic capacity increased (P<0.05) 30±9% and COX IV protein, a marker of mitochondrial content, was elevated (P<0.05) 33±7% after training. Interestingly, aerobic training did not alter the basal gene expression of PGC‐1α and TFAM. However, PGC‐1α protein content was reduced (P<0.05) 20±5% after aerobic training. These data suggest that increased basal levels of PGC‐1α are not necessary for the improvements in whole body and skeletal muscle oxidative capacity with exercise training in elderly women.

Supported by grants NIH (AG032127) and NASA (NJ06HF59G)