bluuebunny/crossref_metadata_2025 huggingface dataset:
Kv1.3 and Kv1.5 Channels as Novel Targets for the Prevention of Restenosis in Human Vessels
Authors: Perez‐Garcia M Teresa , Arevalo‐Martinez Marycarmen , de la Fuente Miguel Angel , Simarro Maria , Alonso Esperanza , Lopez‐Lopez José Ramón , Cidad Pilar
Published: November 2023

The contractile state of vascular smooth muscle cells (VSMCs) in the vessel media determines vascular tone. However, these cells can undergo a phenotypic modulation (PM) towards a synthetic phenotype in response to injury. Upon PM, VSMCs lose their contractile proteins and adquire proliferative, migratory and secretory skills. PM is central to the process of intimal hyperplasia that leads to restenosis of the vessel following vascular injury. We have previously described changes in the expression of Kv1.3 and Kv1.5 channels associated with PM. Proliferating VSMCs show an increased functional expression of Kv1.3 channels with a concomitant loss of Kv1.5 channels. Moreover, Kv1.3 blockers inhibit proliferation and migration in animal models of vascular injury and in VSMCs in primary cultures. In this work, we explored if this Kv1.5 to Kv1.3 swicth is also present in human vessels undergoing PM, and we investigate the underliying mechanisms linking Kv channel switch to the development of intimal hyperplasia.

We explored PM in human mammary artery (hMA) samples using organ culture of arterial rings and primary culture of VSMCs from arterial explants. We tested the effect of Kv1.3 and Kv1.5 manipulations by overexpresion of Kv1.5 with viral vectors and blockade of Kv1.3 channels with the selective inhibitor PAP‐1. The degree of intimal hyperplasia in the organ culture was explored with hystomorphometric analyses. Changes in proliferation, migration and secretion were also studied in both preparations.

Fetal Bovine Serum (FBS) treatment induced intimal hyperplasia leading to stenosis in organ culture, which was prevented with PAP‐1. PAP‐1 decreased both FBS‐induced proliferation and VSMCs secretion, as the increase in the number of elastic laminae induced by FBS was fully reverted in the presence of PAP‐1. Kv1.3 blockers also inhibited proliferation of hMA VSMCs in culture. In addition, overexpression of Kv1.5 channels had an antiproliferative effect that can be explained by Kv1.5 occlusion of Kv1.3 pro‐proliferative effects.

We show that organ culture of human vessels represents a valuable approach for the study of the complex, multifactorial process of vascular restenosis. Many of these processes can be targeted by the use of PAP‐1, so that therapies using Kv1.3 blockers could represent a significant improvement for the prevention of intimal hyperplasia.

Support or Funding Information

Supported by grants ISCIII‐FEDER RD12/0042/0006, (Heracles Program), BFU2013‐45867‐R (MINECO) and by UVA‐Santander fellowship to MAM.
Gender Bias in G Protein‐Coupled Receptor Expression in Human Pulmonary Artery Smooth Muscle Cells
Authors: Iyinikkel Jean , Saunders Fiona , Walker Claire , Murray Fiona
Published: November 2023

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance and proliferation of pulmonary artery smooth muscle cells (PASMC). Female bias of PAH (ratio of 4.1:1, female vs. male) is well known, however not fully understood. G protein‐coupled receptors (GPCRs) are the largest family of receptors and important regulators of PASMC function. Profiling GPCRs in female‐ vs. male‐PASMC could advance the understanding of the disease and highlight the role of novel GPCRs in the female bias of PAH. We used an “unbiased” approach (a TaqMan® GPCR array) to profile GPCR expression in primary human male‐ and female‐PASMC. We found that male‐PASMCs express 190 GPCRs (42 orphan and 148 non‐orphan, n=3), while female‐PASMC express 174 GPCRs (44 orphan and 130 non‐orphan, n=3). GPCR expression correlates with function: for example Gαs‐coupled GPCRs with formation of cAMP and inhibition of cell proliferation, thus documenting that the GPCR array identifies physiologically relevant GPCRs. We found female‐PASMC have an increased expression of 25 GPCRs (>2‐fold) and a decreased expression (>2‐fold) of 19 GPCRs, compared to male‐PASMCs. Chemokine receptor 1 (CCR1) and the purinergic receptor, P2Y12, both of which are Gαi­‐coupled, were highly upregulated in female‐PASMC (21‐ and 62‐fold, respectively). GPR75, an orphan GPCR whose expression was also increased in female‐PASMC (confirmed by real‐time PCR), was up‐regulated by 17β‐estradiol in a dose‐dependent manner (0.1 nM‐1 μM, 24 h). Taken together these data provide evidence that GPCR expression in PASMC is gender specific and such differences may contribute to the physiology of normal PASMC and the female bias of PAH.

Support or Funding Information

ATS foundation and Pulmonary Hypertension Association and University of Aberdeen
Hypoxia‐induced proliferation and migration in pulmonary arterial smooth muscle cells requires β‐catenin.
Authors: Yun Xin , Jiang Haiyang , Shimoda Larissa A
Published: November 2023

Exposure to hypoxia causes proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) in the distal vasculature, leading to structural remodeling of these arteries and contributing to the pathogenesis of pulmonary hypertension. In past studies, we showed that expression of the water channel, aquaporin 1 (AQP1), was upregulated by both in vivo and in vitro exposure to hypoxia, and that this channel mediated PASMC proliferation and migration. The effects of AQP1 were independent of channel water transport, but were associated with increased expression of β‐catenin, a dual function protein that is involved in the Wnt signaling pathway. β‐catenin activates transcription of genes involved in cell growth and survival and regulates the coordination of cell‐cell adhesion. Thus, we explored whether β‐catenin was required for hypoxia‐induced PASMC migration and proliferation. Primary cultures of rat PASMCs were harvested from distal pulmonary arteries and exposed to in vitro hypoxia (4% O2 ‐ 5% CO2) or control conditions. Proliferation and migration were measured using BrdU incorporation and transwell filters, respectively. Exposure to hypoxia for 24–72 h increased PASMC migration and proliferation and β‐catenin protein, but not mRNA, levels. Silencing β‐catenin in PASMCs had no significant effect on cell function under control conditions, but prevented hypoxia‐induced proliferation and migration. Based on our findings, we conclude that increases in β‐catenin protein stability/synthesis, perhaps mediated by AQP1, are crucial for hypoxia‐induced PASMC proliferation and migration.

Support or Funding Information

This work was funded by HL‐07963 and AHA0755479.
Hypoxia Alters Ubiquitination of Proteins Associated with Cellular Proliferation in Mouse Lung
Authors: Zhao Jingru , Wade Brandy , Ma Jing , Hart C. Michael , Sutliff Roy L
Published: November 2023

Pulmonary hypertension (PH) is a chronic condition characterized by increased pressure in the pulmonary vasculature. PH is caused by constriction and/or thickening of arterial walls due in part to cellular proliferation. The underlying mechanisms for PH pathogenesis are not well understood. The ubiquitin proteasome system (UPS) regulates cell growth and protein expression by marking proteins for degradation by attaching one or more ubiquitin moieties to lysines within target proteins. We hypothesized that hypoxia, a common stimulus for PH, would alter the UPS and pathways that regulate pulmonary vascular cell proliferation in PH. The Cell Signaling PTMScan® Ubiquitin Remnant Motif (K‐ɛ‐GG) Kit was used to determine the effect of hypoxia on protein ubiquitination by mass spectrometry (MS). Mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks, and PH was confirmed by measuring right ventricular systolic pressure. Lungs were excised for MS analysis. The MS characterization of ubiquitinated proteins revealed that hypoxia significantly changes (fold change threshold of ≥±1.5) ubiquitination of 198 peptides. Hypoxia significantly increased ubiquitination of 14‐3‐3 eta at a single lysine, and RhoA and AnnexinA2 had increased ubiquitination at two lysines. Hypoxia also significantly decreased ubiquitination of Profilin‐1 at two lysines. These proteins have been studied in other cell types and diseases and are known to regulate cellular proliferation. 14‐3‐3 eta is a chaperone protein that interacts with proteins to promote cellular migration and proliferation in lung cancer cells. RhoA regulates cell division and is downregulated in Group 2 PH. AnnexinA2 expression is increased in hypoxia‐exposed osteoblastic cells, andProfilin‐1 expression is decreased in invasive and metastic breast cancer. We conclude that hypoxia‐induced changes in ubiquitination of 14‐3‐3 eta, RhoA, Profilin‐1, and Annexin A2 may play an important role in PH pathogenesis and regulation of pulmonary vascular cell proliferation in PH.

Support or Funding Information

NIH RO1HL102167 (CMH and RLS), Merit Review from VA Office of Research and Development (1I01BX001910 to CMH), and AHA postdoctoral fellowship (16POST30930007 to BEW), SIRE (to JZ)
The role of microtubule–associated protein EB3 in Pathogenesis of Pulmonary Hypertension.
Authors: Atwal Gurdeep , Sun Mitchell , Yang Xiaoyan , Sun Ying , Chen Jiwang , Komarova Yulia
Published: November 2023

Hypoxia is one of the most powerful triggers of Pulmonary Hypertension (PH) that acts directly on endothelial cells (ECs). Translocation of hypoxia inducible factors (HIFs) from the cytoplasm into the nucleus of ECs plays a critical role in the pathogenesis of PH by inducing transcription of genes containing hypoxia response elements (HREs) such as endothelin1 (ET1), which in turn, promotes vasoconstriction of smooth muscle cells. The role of microtubule (MT) cytoskeleton in the pathogenesis of pulmonary hypertension is not yet understood. Some studies suggest that MT dynamics play a critical role in nuclear translocation of HIFs, and therefore HIF function, but the results of these studies are controversial. Here, we investigated the role of end binding protein (EB3) in endothelial cells in the pathogenesis of PH through stabilization and/or nuclear translocation of HIFs in pulmonary arterial endothelial cells. In order to understand the specific role of EB3 in ECs, we developed tamoxifen‐inducible cell specific knock out (i‐KO) mouse models, allowing deletion of Mapre3 (EB3) gene in either endothelial or smooth muscle cells (SMCs). Exposure of EB3fl/fl (control) or iSMS‐KO mice to 10% hypoxia for four weeks resulted in a significant increase in right systolic ventricular pressure (RSVP) from 22±0.8 mmHg (normoxia) to ~35±0.8 and ~33±1 mmHg, respectively. This increase in RSVP was significantly mitigated in EB3 iEC‐KO mice as compared to the other two groups (p<0.0008). The protective effects of EB3 KO in ECs were even more evident when wall thickness of small arteries was compared between groups (p<0.0001). EB3 KO mice also showed decreased HIF2α immunofluorescence in nuclei of endothelial cells in lungs indicating the critical role of EB3 in HIF2α nuclear transport. Furthermore, our experiments in human pulmonary aorta endothelial cells expressing HIF2α mutants, which are not sensitive to proteolytic degradation in normoxia, conformed the role of EB3 in HIF2α trafficking. We conclude that MT cytoskeleton and EB3 are potential targets to limit HIF function in pathogenesis of PH.

Support or Funding Information

R01 HL103922
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 Information

This 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 Information

Supported 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 Information

Supported 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 Information

Suresh 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 Information

This 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 Information

NHLBI (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 Information

NIH R01HL098200 and R01HL121059, T32GM099608
Differences in tolerance to exertional hyperthermia between male and female mice
Authors: Garcia Christian Kyle , Robinson Gerard Patrick , Mattingly Alex , Laitano Orlando , Van Steenbergen David , King Michelle , Leon Lisa , Clanton Thomas
Published: November 2023

Surveillance reports suggest possible reductions in heat stroke susceptibility in female vs. male active component members of the US Armed Forces, but whether these differences reflect behavior or underlying biology is unknown. Previous studies in mice have shown that females exhibit markedly better resistance to moderate, passive heat. However, whether heat tolerance translates to acute settings or to exertional heat stroke (EHS) has not been tested. In this study, we compared responses in male and female mice to an established model of EHS. Exercise trained mice (3 wks) were maintained at 37.5°C (35% RH) and ran using a preprogrammed incremental protocol on a forced running wheel. The EHS end point was defined as loss of consciousness. Female mice on average ran longer than males (177 vs. 124 min; p=0.0001), and were exposed to greater heat loads (241 vs. 160 °C • min; p=0.0001). Male and female mice ran to nearly identical average peak core temperatures, both 42.2°C (n.s.). There were no differences in the minimum temperature during post EHS hypothermia 32°C (n.s.) or the time to reach the minimum temperature. However, females lost a greater % body weight (9.2% vs 7.5% p < 0.001), demonstrated significantly higher levels of circulating corticosterone (286 vs 183 ng/ml, p = 0.001, 3 h) and higher levels of resistin polypeptide (8891 vs. 3781 pg/ml, p = 0.004, 3 h). These results demonstrate that female mice have greater resistance to EHS during exercise in hyperthermia. Possible mechanisms include greater body surface to mass ratio in females vs. males (3.3 vs. 3.2 m2/kg; p=0.0001), greater aerobic conditioning in females (characteristic of mice), or a hormonally or genetically induced resistance to hyperthermia. Though controversial, marked elevations in circulating corticosterone and resistin in females have the capacity to contribute to improved heat tolerance. We conclude that female mice are significantly more resistant to EHS than male mice. Inherent thermal tolerance in female mice may provide an evolutionary advantage because metabolic rate and heat production have been shown to double during pregnancy and lactation. Author views not official US Army or DoD policy. W81XWH‐15‐2‐0038
Whole‐body heat stress does not appear to reduce β1‐adrenergic responsiveness in heat stressed older individuals
Authors: Moralez Gilbert , Kouda Ken , Hieda Michinari , Hardin Elizabeth , Sarma Satyam , Romero Steven A , Cramer Matthew N , Gagnon Daniel , Adams Amy N. , Jaffery Manall F , Crandall Craig G
Published: November 2023

Whole‐body heat stress increases cardiac output in younger and older individuals (i.e. ≥60 years), though the magnitude of that increase is attenuated in the older group. Recently, it has been identified that attenuated increases in cardiac output in older individuals during heat stress is primarily due to diminished heart rate responses. However, the mechanisms by which the increases in heart rate are attenuated in heat‐stressed older individuals remains unknown. The purpose of this study is to test the hypothesis that attenuated β1‐adrenergic responsiveness contributes to the lower increase in heart rate observed in older adults during heat stress. In ten healthy young (five females: age 23 ± 2 and five males: age 26 ± 3) and four older participants (two females: age 63 ± 2 and two males: age 70 ± 2) heart rate was measured prior to and during intravenous infusion, of a low (5 μg/kg/min) and a moderate (15 μg/kg/min) dose of dobutamine (a β1‐adrenergic receptor agonist) under normothermia (NT) and hyperthermia (HT; increase in internal temperature of 1.2 ± 0.1°C). The heart rate response to heat stress alone tended to be attenuated in older individuals (+24 ± 3 older: +33 ± 2 beats/min young, P = 0.06). The low dose of dobutamine during HT further increased HR in the older (Δ 23 ± 7 bpm) and young participants (Δ 12 ± 5 bpm) to a similar extent (older vs. young; P=0.15). Likewise, the infusion of the high dose of dobutamine during HT similarly increased HR in both groups (Δ 45 ± 8 vs. young: Δ 42 ± 5 beats/min, P = 0.80). Based upon these preliminary data, healthy aging does not reduce β1‐adrenergic responsiveness under normothermic and heat stress conditions. This finding suggests that attenuated increases in heart rate during heat stress in older individuals are likely neurally mediated.
Prior Infection and Prior Heat Illness as Risk Factors for Exertional Heat Stroke
Authors: King Michelle A. , Ward Matthew D. , Gabrial Charles H. , Audet Gerald N. , Adams Bruce , Leon Lisa R
Published: November 2023

Anecdotal evidence suggests that incidence of exertional heat stroke (EHS) is higher in individuals that have experienced prior infection, prior heat illness, or rhabdomyolysis; however these hypotheses are lacking in scientific validation. To examine these relationships, we searched the Medical Health System Data Repository (MDR) for active duty service members (ADSM) who had at least one EHS during the years of 2008–2014. Incidence was identified using the International Classification of Disease 9th (ICD‐9) Revision codes from de‐identified electronic medical records. Over this time period, 2,529 ADSM experienced at least one episode of EHS, while 12% of these individuals experienced multiple events (≥2). Within one year preceding EHS, 20% of ADSM suffered a prior heat illness and 9% were diagnosed with rhabdomyolysis. Individuals that suffered a prior heat injury (HI) were 16 times more likely to develop rhabdomyolysis (OR 16.07; 95% CI (11.32–22.81)). Thirty percent of ADSM were also diagnosed with at least one prior infection in the two months preceding EHS or in the seven days following the HI. Further, these individuals were more likely to have suffered a prior HI (OR 1.35; 95% CI (1.03–1.78)). Current medications, immunizations, and allergy diagnosis were then examined as risk factors (RF) for increased susceptibility to EHS. Prescriptions for anti‐inflammatories, anti‐infection, and central nervous system agents were prescribed most often, with 33% of individuals taking at least one medication in the two week time period before EHS. Individuals prescribed anti‐inflammatories (15%) were more likely to have experienced a prior HI (OR 0.36; 95% CI (0.24–0.55)). Interestingly, in the 30 day time period before EHS, 20% of individuals received at least one immunization. Unexpectedly, allergy diagnosis was identified as a risk factor for EHS, occurring in 19.5% of EHS patients. Episode length (EL) (time from clinical admission to release) was used as a gauge of EHS severity. Both prior HI and rhabdomyolysis extended EL (P<.001). Examination of specific organ failure(s) was used to identify tissues most compromised by EHS. Renal failure was most commonly reported, appearing in 30% of EHS cases. Known RFs for EHS such as time of year, elevated body mass index, and advanced age were confirmed. This epidemiological review defines the link between EHS and prior infection, prior heat illness, and rhabdomyolysis and highlighted other lesser known RFs such as immunizations and allergies in ADSM. Author view not official US ARMY or DoD policy.
Indicators of kidney ischemia/reperfusion injury predict heat stroke severity in a conscious rodent model
Authors: Ward Jermaine Allen , Dineen Shauna , Plamper Mark , Leon Lisa , Audet Gerald
Published: November 2023

Currently there are no specific biological markers capable of differentiating between levels of heatstroke (HS) severity. Ischemia/Reperfusion (I/R) is a known concurrent injury pathway that occurs with HS. I/R is a well‐studied condition, which we believe could be leveraged for the discovery of novel HS severity biomarkers. The renal system is one of the critical organ systems in HS pathophysiology. In the current study we investigated whether five well‐established markers of I/R injury in the kidney could also serve as indicators of systemic HS severity level. In this study radiotelemetry implanted Fischer 344 rats were passively heated to a core temperature (Tc) of 42.0±0.1˚C. Their blood pressure (BP) and heart rate (HR) were also monitored continuously during heating and recovery. The rats were then sacrificed at 24 hrs, at which point tissue and blood samples were collected. Combining physiological data (Tc, HR, BP) and histological analysis of tissues, heat exposed rats were categorized into three groups; mild, moderate and severe. Intercellular adhesion molecule 1 (ICAM‐1), Urea, Creatinine, Endothelin‐1 and Lipocalin were analyzed in the kidney and/or plasma for their efficacy as HS severity markers. The latter three markers showed no significant changes across groups in kidney tissue or plasma. ICAM‐1 concentration in kidney tissues showed a significant rise in the moderate (1150.26±70.47 pg/ml, p<0.01) and severe (1155.66±119.85 pg/ml, p<0.03) groups when compare to the controls (816.88±76.21 pg/ml). Rats in the mild group were not significantly changed from the controls (1127.31±133.88, p<0.06) but did show a strong trend for an increase in ICAM‐1 concentration. Urea levels in the plasma parallel robustly with increases in ICAM‐1 in the kidney, and show a distinct rise in severe (93.380±29.19 mg/dL, p<0.007) and moderate (91.733±20.49 mg/dL, p<0.010) HS populations compared with controls (36.916±1.05 mg/dL). Urea levels in the mild group showed comparable levels as the controls (37.422±2.25 mg/dL, p<0.832). In conclusion, the increase in ICAM‐1 and urea suggest significant I/R injury, while also successfully differentiating between levels of HS severity, demarcating mild HS animals from moderate and severe. With further investigation, these results may lead to improved diagnosis and treatment of HS patients. Author views not official US Army or DoD policy.
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

OBJECTIVE

Ketogenic 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.

METHODS

Male 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.

RESULTS

Weights 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).

CONCLUSION

The 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 Information

This study was funded in part by a BBSRC New Investigator Award BB/L023547/1 to AP
Effect of Resveratrol as Caloric Restriction Mimetic and Environmental Enrichment on Body Weight, Lipid Profile and Electrolytes in Young Healthy Mice
Authors: Muhammad Mustapha Shehu , Magaji Rabiu Abdussalam , Mohammed Aliyu , Isa Ahmed‐Sherif , Garba Magaji Mohammed , Hassan Adamu
Published: November 2023

Caloric restriction (CR) and Environmental Enrichment have been separately reported to possess health benefits such as protection against obesity, cancer, cardiovascular and neurodegenerative diseases. Resveratrol, a natural polyphenolic compound has been reported to be caloric restriction mimetic. The aim of this study was to investigate the potential benefit of the combination of Resveratrol as CR mimetic and EE on Lipid Profile in young healthy mice. Fifty mice of both sexes were randomly divided into five groups of 10 animals each: group I served as control, group II were maintained on alternate day feeding (ADF), group III received Resveratrol 50 mg/kg, suspended in 10 g/L of (CMC) orally per kg/day. Group IV received CMC and kept in an Enriched Environment (EE) while group V received Resveratrol 50 mg/kg and kept in EE. The treatment lasted for 28 days. The animals were sacrificed 24 hours after the last treatment and the plasma sample was collected for biochemical evaluation. The results obtained showed significant decrease (P < 0.05) in high density lipoprotein concentration in ALDF group when compared to the control. A significant increase (P < 0.05) was also observed in low density lipoprotein concentration in ADF group when compared to the control. There was no significant change observed in the body weight and electrolyte contents of the animals during the study period. In conclusion, the results obtained indicates that Resveratrol as CR mimetic and EE have no significant effects on body weight, lipid profile and electrolyte contents in young healthy mice over a period of four weeks.

Effect of Resveratrol‐induced CR and EE on lipid profiles in mice

Groups
TC (mg/dl)
TG (mg/dl)
HDL (mg/dl)
LDL (mg/dl)

Control (CMC)
2.38 ± 0.10
0.86 ± 0.08
1.088 ± 0.08
1.12 ± 0.05

ALDF
2.45 ± 0.07
0.94 ± 0.07
0.70 ± 0.06*
1.56 ± 0.10*

Resveratrol only
2.23 ± 0.07
0.88 ± 0.10
0.75 ± 0.08
1.30 ± 0.07

EE only
2.38 ± 0.08
0.96 ± 0.05
0.76 ± 0.10
1.48 ± 0.16

Resveratrol + EE
2.51 ± 07
0.80 ± 0.09
0.87 ± 0.09
1.48 ± 0.08

P<0.05,

CMC = Carboxymethyl‐cellulose, ALDF = Alternate day feeding, EE = Environmental Triacylglycerol, HDL = High density lipoprotein, LDL = Low density lipoprotein

Effect of Resveratrol induced CR and EE on Electrolyte content in mice

Groups
Na (mmol/L)
K (mmol/L)
Cl (mmol/L)

Control (CMC)
139.75 ± 0.92
4.04 ± 0.11
99.75 ± 1.88

EODF
139.30 ± 0.86
4.17 ± 0.11
100.70 ± 0.93

Resveratrol only
129.60 ± 10.42
4.02 ± 0.15
100.70 ± 1.13

EE only
139.00 ± 1.21
4.11 ± 0.18
100.13 ± 1.29

Resveratrol + EE
137.30 ± 1.28
4.33 ± 0.09
99.60 ± 1.09

* P < 0.05,

CMC = Carboxymethyl‐cellulose, ALDF = Alternate day feeding, EE = Environmental enrichment, Na = Sodium, K = Potassium, Cl = Chloride
Molecular mechanism of cytokinesis
Authors: Pollard Thomas D.
Published: November 2023

We use three complementary approaches to study the molecular basis of cytokinesis. We characterize the structures and biophysical properties the participating proteins to generate hypotheses about the biochemical reactions. Quantitative fluorescence microscopy of live cells expressing fluorescent fusion proteins gives the numbers of each type of protein in whole yeast cells with one second temporal resolution. High‐speed super resolution localization microscopy (FPALM) allows us to image structures at 35 nm resolution in live cells on a time scale of a few seconds. Comparisons of these quantitative measurements in cells with simulations of molecularly explicit mathematical models of the reactions in our biochemical hypothesis usually suggest ways to improve our hypotheses. Our simulations now reproduce the time course of events during the assembly and constriction of the cytokinetic contractile ring as observed in live cells and predict the outcomes of experimental manipulations.

Support or Funding Information

NIH research grant GM026132