bluuebunny/crossref_metadata_2025_split huggingface dataset:
AGE/RAGE Signaling in Diabetes‐Mediated Vascular Calcification in Vascular Smooth Muscle Cells
Authors: Kay Amber M. , Rushing B. J. , Simpson C. L. , Stewart J. A. 
Published: November 2023

Advanced Glycation End‐Products (AGEs)/Receptor for AGEs (RAGE) signaling has been a well‐studied cascade in different pathologies, particularly type II diabetes mellitus. The complex nature of the receptor, ligand specificity, and intersecting pathways of AGE/RAGE signaling is still not well understood. AGE/RAGE signaling influences both cellular and systemic responses to increase bone matrix proteins through p38 MAPK and ERK1/2 signaling pathways in hyperglycemic and calcification conditions. AGE/RAGE signaling has also been shown to increase oxidative stress by promoting diabetes‐mediated vascular calcification through NOX‐1 activation and decreased SOD‐2 expression to promote a phenotypic switch of vascular smooth muscle cells (VSMCs) to osteoblast‐like cells. The purpose of this research is understand AGE/RAGE mediated vascular calcification as a complication of diabetes. Calcification was induced in primary mouse VSMCs of non‐diabetic (HetRWT), diabetic (db/dbRWT), non‐diabetic RAGE knockout (HetRKO), and diabetic RAGE knockout (db/dbRKO), and then subsequently treated with AGEs to activate RAGE. Intracellular calcium levels were quantified and showed a pronounced calcification in db/dbRWT and loss of RAGE resulted in a decrease in calcification in db/dbRKO. Western blotting analysis revealed VSMC marker protein (α‐smooth muscle actin) was decreased in db/dbRWT calcified cells indicating an osteoblast‐like phenotypic switch and this was not observed in db/dbRKO VSMCs. These data demonstrated that RAGE has a role in diabetes‐mediated vascular calcification. By understanding the role, the AGE/RAGE signaling cascade plays diabetes‐mediated vascular calcification will allow for possible targets for pharmacological intervention to be identified that may decrease the severity of this diabetic complication.Support or Funding InformationThis research was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 2015202674. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Also, it was supported by the American Heart Association Scientist Development Grant (11SDG5310006). NIH Academic Research Enhancement Award (R15)(PA‐13‐313), and Department of Defense Peer Reviewed Medical Research Program ‐ Investigator‐Initiated Research Award PR150906 ‐ GRANT12018632.
Increasing resting tension enhances cGMP production in normotensive and hypertensive rat aortas
Authors: Silva Bruno R , Grando Marcella Daruge , Bendhack Lusiane Maria 
Published: November 2023

NO‐Synthase (NOS) is a Ca2+‐Calmodulin‐dependent enzyme that is activated by phosphorylation of Ser1177 (P‐Ser) producing NO and inactivated by phosphorylation of its site Thr495 (P‐Thr). The main target in NO signaling is soluble guanylyl‐cyclase (sGC) and the production of cGMP. Shear stress‐mediated phosphorylation of NOS regulates NOS activity. However, the signaling in renal hypertensive (2K‐1C) rat aorta submitted to increasing resting tension (RT) is not known. Our hypothesis is that RT can activate the endothelial NOS (eNOS) increasing NO production and consequently cGMP in normotensive (2K) and 2K‐1C rat aortas. This study aimed to evaluate the effects of RT of 1.5 g (control) and 3.0 g on the eNOS phosphorylation in phenylephrine (PE)‐induced contractile response in isolated aorta from 2K‐1C and 2K rats. Concentration‐effect curves for PE and for extracellular CaCl2 were constructed in aortas under resting tension of 1.5 g and 3.0 g in the absence or presence of NOS inhibitor (L‐NAME, 100 μM). The expression of P‐Ser and P‐Thr were evaluated by Western Blot. The cGMP was measured by ELISA‐Immunoassay. This study was performed in accordance with the Ethics Committee CEUA‐USP (15.1.477.60.1). PE‐induced maximum contractile effect (ME) on RT of 1.5 g was lower in 2K‐1C (1.2 ± 0.2 g, n=7; p<0.001) than in normotensive 2K (2.2 ± 0.1g; n=5), which effects were normalized by NOS inhibition with L‐NAME. PE‐induced contractile response was similar between 2K‐1C and 2K under RT of 3.0 g. Similar responses were obtained for CaCl2. However, NOS inhibition increased the contractile response induced by CaCl2 in RT‐dependent way only in 2K‐1C (1.5 g: 0.72 ± 0.09 g; 3.0 g: 1.03 ± 0.10 g; n=4; p<0.05). P‐Ser1177 expression stimulated by PE was higher in 2K‐1C than in 2K aortas. Increasing of RT from 1.5 g to 3.0 g decreased P‐Ser and increased P‐Thr in 2K aorta, but it was not altered in 2K‐1C. Increasing RT from 1.5 g to 3.0 g increased cGMP production in 2K (1.5 g: 4.4 ± 0.7 pmol/ml; 3.0 g: 7.1 ± 1.0 pmol/ml; n=5; p<0.05) and 2K‐1C (1.5 g: 2.5 ± 0.5 pmol/ml; 3.0 g: 4.9 ± 0.5 pmol/ml; n=5; p<0.05), which was higher in 2K (p<0.05) than in 2K‐1C. Taken together, our results demonstrate that the anti‐contractile effect induced by PE in 2K‐1C rat aortas under 1.5 g tension is due to NOS activation by calcium transient and higher P‐Ser1177. The anti‐contractile effect induced by PE in 2K‐1C aorta is abolished by increasing RT to 3.0 g. The increased RT in 2K‐1C aortas to 3.0 g also increases cGMP production despite of this production to stay lower than in 2K rat aortas.Support or Funding InformationSupported by FAPESP, CNPq and CAPES
Rho‐associated kinase and zipper‐interacting protein kinase: roles in rapid phosphorylation events in serum‐stimulated human arterial smooth muscle cells
Authors: Walsh Michael P. , Deng Jing‐Ti 
Published: November 2023

Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Rho‐associated kinase (ROCK) and zipper‐interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par‐4 (prostate‐apoptosis response‐4). Phosphorylation of MYPT1 at T696 and T850 inhibits MLCP activity whereas phosphorylation of Par‐4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of ROCK and ZIPK in these rapid serum‐induced phosphorylation events, we investigated the time‐courses of phosphorylation of LC20, MYPT1 and Par‐4 in serum‐stimulated human vascular smooth muscle cells, and examined the effects of siRNA‐mediated ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T850, and Par‐4 at T163, peaking within 30–120 s. ROCK1 knockdown decreased the levels of phosphorylation of LC20, MYPT1 and Par‐4, whereas ZIPK knockdown decreased LC20 phosphorylation, but increased phosphorylation of MYPT1 and Par‐4. Consistent with the effects of kinase knockdown, ROCK inhibition by GSK429286A and H1152 reduced serum‐induced LC20, MYPT1 and Par‐4 phosphorylation, while ZIPK inhibition by HS38 reduced only LC20 phosphorylation. We conclude that (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1 and Par‐4, (ii) both ROCK and ZIPK are involved in LC20 phosphorylation, and (iii) only ROCK accounts for MYPT1 and Par‐4 phosphorylation.Support or Funding InformationSupported by a grant from the Canadian Institutes of Health Research (MOP‐111262).
Exercise ameliorates the development of diabetic retinopathy
Authors: Homme Rubens Petit , Metreveli Naira , Veeranki Sudhakar , Tyagi Suresh 
Published: November 2023

One of the symptoms caused by Diabetic Retinopathy (DR) include leaky blood vessels, which causes a distortion in vision. DR generally manifests itself as asymptomatic on its onset, and is usually diagnosed in its proliferative phase. At this stage, therapeutic efforts are generally ineffective. Additionally, current therapeutic methods are palliative and not curative. Recent studies have suggested a causative role for metalloproteinase‐9 (MMP‐9) in DR through (1) facilitating the apoptosis of retinal capillaries cells in the early development of the disease and (2) contributing to the abnormal neovascularization in the latter stages of the disease. Other studies have indicated that exercise reduces the level of MMP‐9 in plasma, as well as reducing its level in the heart of patients with Type II Diabetes. We hypothesize that exercise ameliorates the development of diabetic retinopathy through suppressing the activity and level of MMP‐9. The level, activity and mRNA expression of MMP‐9, vascular endothelial growth factor (VEGF), and corneal collagen content in diabetic mice (db/db) were measured. Retinal vasculature tree morphology was also analyzed for apoptosis of retinal capillary cells. Fluorescence angiography was performed as well to analyze the leakiness of retinal vessels. The data resulted from the retinal vascular tree morphology suggested aneurysmic‐extreme dilation, which weakens the walls of retinal blood vessels‐and acellular capillaries in db/db mice, exercises reduces these defects. Further analyses should indicate that exercise reduces the defects in the retinal vasculature of db/db mice.Support or Funding InformationSuresh Tyagi, PhD
Intravascular Pressure Self‐Regulates the Myogenic Response by Controlling Surface PKD2 (TRPP1) Channel Abundance in Smooth Muscle Cells of Skeletal Muscle Arteries
Authors: Hasan Raquibul , Bulley Simon , Jaggar Jonathan H. 
Published: November 2023

Intravascular pressure stimulates arterial smooth muscle cell depolarization, leading to voltage‐dependent Ca2+ channel (Cav1.2) activation and vasoconstriction; a reaction termed the ‘myogenic response’. PKD2 (also termed TRPP1), is a non‐selective cation channel, expression of which has been demonstrated in myocytes of cerebral and mesenteric arteries. Mechanisms that regulate PKD2 activity are unclear. Here, we investigated the regulation of PKD2 surface abundance by intravascular pressure in murine hindlimb arteries using a combination of Western blotting, arterial biotinylation, and pressurized artery myography techniques. We found that PKD2 is primarily plasma membrane‐localized in myocytes of hindlimb arteries. Loss of intravascular pressure due to depressurization lead to PKD2 internalization and lysosomal degradation. In contrast, arterial depressurization did not alter TRPM4, TRPC6, CaV1.2, ANO1, or KV2.1 proteins. Intravascular pressure (80 mmHg) or a bath solution containing 30 mM K+, which depolarizes arteries to physiological membrane potential, prevented PKD2 internalization and degradation. Nimodipine, a voltage‐dependent Ca2+ channel (Cav1.2) blocker, inhibited depolarization‐induced stabilization of PKD2 protein. PKD2 surface protein and myogenic tone were measured in arteries pressurized to either 10 or 80 mmHg. Pressurizing arteries to 80 mmHg prevented the loss of both surface PKD2 and myogenic tone that occurred in arteries pressurized to 10 mmHg. Consistent with a central function of PKD2, myogenic tone was strongly attenuated in hindlimb arteries from conditional, smooth muscle‐specific PKD2 knockout mice. In summary, we show that intravascular pressure promotes PKD2 surface expression in arterial smooth muscle cells through a mechanism involving membrane depolarization and Cav1.2 channel activation, to cause vasoconstriction. Thus, intravascular pressure self‐regulates the myogenic response by controlling PKD2 surface abundance in arterial smooth muscle cells.Support or Funding InformationThis study was supported by NIH/NHLBI grants to J.H.J. and American Heart Association grants to R.H. and S.B.
Changes in Vascular Function with Aging in Fibrillin‐Mutant Mice
Authors: Pham Y. Hong My , Roos Carolyn M , Smith Leslie A , Zhang Bin M , Verzosa Grace C , Hagler Michael A , Thalji Nassir M , Miller Jordan D 
Published: November 2023

In Marfan syndrome, mutations in the fibrillin gene are known to result in aortic dilatation and aneurysm formation, which is associated with impaired endothelial function and increased vascular stiffness. Previous studies suggested these phenotypes may be partly attributable to overactivation of TGF β signaling, which leads to increased reactive oxygen species production, extracellular matrix production, and increased matrix metalloproteinase activity in aortic wall. To our knowledge, however, there are no studies comprehensively evaluating the relationships between conduit vessel vasomotor function, vascular stiffness, and aortic dilatation in young and aged models of Marfan syndrome. Thus, the aim of this study was to test the hypothesis that carotid arterial vasomotor dysfunction and increased vascular stiffness precede aortic dilatation in fibrillin mutant mice. We used wild‐type (+/+) or fibrillin mutant (+/C1037G) mice fed a normal chow diet for 3 months (young) or 14 months (old), measured changes in vasomotor function and vascular stiffness in carotid arterial segments using pressurized arteriography, aortic dimensions using high resolution echocardiography, gene expression using qRT‐PCR, and protein levels using immunohistochemistry. Consistent with our hypothesis, young +/C1037G mice had impaired endothelium‐dependent relaxation to acetylcholine (ACH) compared to +/+ littermates, but did not exhibit changes in vascular stiffness. In contrast, old +/C1037G mice did not have significant changes in vasomotor responses to ACH compared to +/+ mice, but displayed markedly increased vascular stiffness compared to their wild‐type littermates. Aortic dimensions were significantly increased in young +/C1037G mice compared to their +/+ littermates, and these changes were exaggerated further with aging. Consistent with previous reports, we found increased TGF beta‐1 expression and canonical TGF beta signaling (p‐Smad2/3 levels) in +/C1037G mice. Interestingly, we also observed increased expression of Runx2 with aging in +/+ mice with aging, and expression of Runx2 was substantially increased in +/C1037G mice compared to their +/+ littermates at both time points. Collectively, our data show that vasomotor function and vascular stiffness in carotid arterial segments change independent of changes in aortic root diameter, suggesting significant heterogeneity in the mechanical and molecular responses to fibrillin deficiency throughout the vascular system.Support or Funding InformationNHLBI (R01‐111121)
A PKA‐coupled purinergic receptor macromolecular complex anchored by AKAP150 potentiates L‐type Ca2+ channel activity and vasoconstriction in diabetic hyperglycemia
Authors: Prada Maria Paz , Syed Arsalan U. , Nystoriak Matthew A. , Patriarchi Tommaso , Nieves‐Cintrón Madeline , Hell Johannes W. , Navedo Manuel F. 
Published: November 2023

Enhanced contractility of vascular smooth muscle in response to elevated extracellular glucose (HG), the hallmark metabolic abnormality in diabetes, contributes to vascular dysfunction associated with cardiovascular complications, such as hypertension. We previously showed that activation of the cAMP‐dependent protein kinase (PKA) anchored by the scaffolding protein AKAP150 has been associated with potentiation of vascular L‐type CaV1.2 channel (LTCC) activity in vascular smooth muscle (VSM) during HG. Moreover, we found evidence implicating a nucleotide‐activated purinergic receptor as a key player in the PKA‐mediated potentiation of LTCC activity and increased vasoconstriction during HG. Here, we tested the hypothesis that a vascular purinergic receptor coupled to Gs exists in a macromolecular complex with AKAP150, PKA, and LTCCs that is necessary for PKA‐mediated LTCC stimulation and enhanced vasoconstriction by HG. Super resolution microscopy, proximity ligation assays, and co‐immunoprecipitation revealed that a purinergic receptor coupled to Gs associate and co‐localize with AKAP150 and LTCCs in wild type and human VSM. Using patch clamp electrophysiology and pressurized cerebral arterial diameter measurements we found that the increase in LTCC current and vasoconstriction in response to HG and a specific activator of this purinergic receptor were ameliorated in VSM from AKAP150 knock out mice. Altogether, our findings suggest a critical role for an AKAP150‐anchored macromolecular complex formed by a Gs‐coupled purinergic receptor, PKA and LTCC in PKA‐mediated stimulation of LTCC activity and vasoconstriction during HG.Support or Funding InformationNIH R01HL098200 and R01HL121059, T32GM099608
The Effects of a Long‐Term Ketogenic Diet on Molecular Markers of Cardiac Hypertrophy
Authors: Romero Matthew Anastacio , Hyatt Hayden W. , Kephart Wesley C. , Mumford Petey W. , Lowery Ryan P. , Wilson Jacob M. , Roberts Michael D. , Kavazis Andreas 
Published: November 2023

OBJECTIVEKetogenic diets (KD) have been investigated for their ability to alter body composition as well as a possible nutritional intervention for epilepsy; however, little is known about long‐term adherence to a KD and its physiological impact. Considering the fat content of a KD and recent research that show a high fat diet may cause cardiac hypertrophy, the objective of this study was to investigate the possible positive or negative responses from long‐term adherence to a KD in cardiac muscle.METHODSMale Fisher rats (4 mo. old) were provided isocaloric amounts of KD (5.2 kcal/g, 23.1% protein, 9.6% carbohydrate, and 65.3% fat, n=6) or standard chow (SC) (3.1 kcal/g, 24% protein, 58% carbohydrate, 18% fat; n=6) for 8 months.RESULTSWeights between groups were lower in the KD group compared to SC (p<0.01), while overall heart weights were not statistically different (p=0.35). Interestingly, heart weights relative to body weight did change with the KD group having higher relative heart weight compared to SC (p<0.01). To better characterize the molecular signals of cardiac hypertrophy we investigated markers for ribosome biogenesis. Factors involved in the transcription of ribosomal DNA (rDNA), RNA polymerase 1 and c‐Myc were not different between groups (p=0.97 and p=0.81), however, upstream binding factor (UBF) was increased in the SC group compared to KD (p=0.01). We also investigated markers of protein synthesis. Eukaryotic translation initiation factor 4E (EIF4E) was not statistically different between groups (p=0.41). Phosphorylated 4E binding protein 1 (4EBP1), the binding protein of EIF4E, was not statistically different between groups (p=0.38) nor was pan‐4EBP1 (p=0.93) along with the ratio of phosphorylated/pan 4EBP1 (p=0.54). Ribosomal protein L5 (RPL5) was not different between groups. Phosphorylated ribosomal protein S6 (RPS6) was higher in the SC groups (p=0.05), while pan‐RPS6 was not significantly different (p=0.70), however, phosphorylated/pan RPS6 was not statistically different between groups (p=0.14).CONCLUSIONThe KD group did show a decrease in body weight, however, heart weights were not statistically different. although some molecular markers of ribosome biogenesis and protein synthesis were higher in the SC group. Future studies are needed to better characterize these molecular markers on cardiac hypertrophy.
The Effect of Graded Skeletal Muscle Glycogen Depletion on Whole‐body and Skeletal Muscle Metabolic Regulation
Authors: Dent Jessica R , Stocks Ben , Ogden Henry , Zemp Martina , Philp Andrew 
Published: November 2023

Exercise training repeatedly exposes skeletal muscle to cyclical periods of glycogen depletion and re‐synthesis. There is evidence to suggest that signal transduction pathways central in driving exercise adaptation are enhanced when exercise is performed in a glycogen depletion state. The purpose of the current study was to use exercise and carbohydrate re‐feeding strategies to manipulate glycogen content in skeletal muscle, allowing examination as to whether a critical level of glycogen depletion is required to promote whole body and skeletal muscle metabolic adaptation. Eight recreationally active males (mean ± SD: age 25 ± 2 yrs; height 176.3 ± 7.7 cm; body mass 78.7 ± 12.8 kg; VO2peak 45.6 ± 4.2 ml·kg‐1·min‐1) each completed three trials in a randomized crossover design. On each occasion, participants undertook an evening bout of muscle glycogen depleting cycle ergometry (~4–6 pm, intervals of 90, 80, 70 or 60 % peak power output (PPO) alternated with recovery intervals at 50% PPO), before consuming carbohydrate (CHO) jelly and beverages at rates of: 0.0 g.kg.h‐1 (LOW), 0.6 g.kg.h‐1 (MED) or 1.2 g.kg.h‐1 (HIG) for 7 hours before returning to the laboratory the following morning (~8 am) after an overnight fast to perform 1 h steady‐state (SS) cycling at 57% PPO. Skeletal muscle biopsies were obtained immediately prior to glycogen depleting exercise (‘resting muscle glycogen’) during trial 2 and pre‐ and immediately‐post each SS exercise session. Three minute expired gas samples were collected prior to and every 15 min during SS exercise to determine O2, CO2, ventilatory exchange (VE), respiratory exchange ratio (RER), CHO (CHO‐ox) and fat oxidation (fat‐ox). The exercise and nutritional model successfully achieved three statistically (p < 0.05) ‘graded’ levels of pre‐exercise glycogen: LOW: 194.6 ± 52.3 μmol·g‐1·dw, MED: 351.7 ± 68.6 μmol·g‐1·dw, HIGH: 475.3 ± 43.9 μmol·g‐1·dw (representing 37.9 ± 8.9, 69.8 ± 15.4 and 94.1 ± 12.5 % of ‘normal resting’ stores: 511.6 ± 69.8 μmol·g‐1·dw). During SS exercise muscle glycogen decreased to a comparable extent in each trial (p < 0.01), however, by the end of exercise concentrations were similar between the MED and HIG trials (p > 0.05). After 15 min of SS exercise, three levels of CHO‐ox and two levels of fat‐ox (MED and HIGH similar) were evident (p < 0.05). As SS exercise continued RER and CHO‐ox were lower (p < 0.05) and fat‐ox greater at every time point in LOW compared to the HIG trial (p < 0.05), while substrate utilization was similar between MED and HIG trials for the remainder of exercise. In conclusion, we provide evidence of a working model to assess the role of skeletal muscle glycogen content on resting and exercise‐induced signaling responses in human skeletal muscle.Support or Funding InformationThis study was funded in part by a BBSRC New Investigator Award BB/L023547/1 to AP