IntroductionHypertension and impaired tissue perfusion are frequent comorbidities in obesity. Since resistance arteries are the primary regulators of peripheral resistance and hence, systemic blood pressure and local blood flow control, we hypothesized that resistance arteries isolated from obese mice would display augmented myogenic reactivity and altered vasomotor responses, compared to non-obese controls. MethodsEight-week-old C57BL/6J mice were fed either a high-fat diet (60% calories from fat; HFD) or a matched control diet for 16 weeks. Body weight, fasting blood glucose, oral glucose tolerance and insulin tolerance were measured. In parallel studies, we measured mean arterial pressure, conducted echocardiographic measurements of cardiac morphology and function and assessed skeletal muscle, mesenteric and cerebral resistance artery reactivity ex vivo with pressure myography. ResultsHFD mice exhibited substantial weight gain and metabolic dysfunction compared to controls. Left ventricular wall thickness and mass were increased in HFD mice, but no other morphological or functional cardiac parameters were different from controls. Blood pressure was modestly increased in HFD mice (from 81 to 87 mmHg; measured under anesthesia); however, contrary to our hypothesis, resistance arteries from HFD mice showed no overt microvascular phenotype in any microvascular bed tested (i.e., no differences in passive diameter, myogenic reactivity or vasomotor responses to phenylephrine or acetylcholine). ConclusionWe conclude that resistance artery function is unaltered in this diet-induced model of obesity with metabolic dysfunction.

Currently there is no effective pharmacotherapy to prevent the growth and rupture of abdominal aortic aneurysms. Using a mouse model that combines cigarette smoke exposure and hypercholesterolemia, we demonstrated that cigarette smoke exacerbated atherosclerosis, leading to elastin fragmentation, aneurysm formation, rupture and death. Arterial injury was driven by macrophages that accumulated within atherosclerotic plaques and exhibited tissue-degrading proteolytic activity in vivo (a process dependent on the endothelial cell-derived macrophage growth factor CSF-1). Single-nucleus RNA sequencing revealed that cigarette smoke-induced endothelial cell dysfunction promoted monocyte recruitment and inflammatory signaling and amplified vascular injury. Furthermore, single-cell transcriptomic analysis identified conserved macrophage responses across mouse and human abdominal aortic aneurysm, including TREM2+ macrophages, which were key mediators of arterial damage. These findings established atherosclerotic plaque macrophages as critical drivers of aneurysm pathology and provide key insights into the mechanisms underlying aneurysm progression and rupture.

Right ventricular (RV) pressure and volume loading induce RV fibrosis in association with RV dysfunction, morbidity, and mortality in repaired tetralogy of Fallot. Transforming‐growth factor‐β1 (TGFβ1) and platelet‐derived growth factor (PDGF) activate common downstream signaling pathways via TGFβ canonical and non‐canonical signaling to promote increased fibroblast activation, proliferation, and fibrosis in other organs. However, the role of PDGF and TGFβ canonical and non‐canonical signaling in RV fibrosis is incompletely characterized. Here, we investigate whether dual inhibition of TGFβ and PDGF, using Tranilast (TRN), improves RV remodeling in response to pulmonary artery banding (PAB) or pulmonary regurgitation (PR). TRN reduced TGFβ canonical signaling in PAB rats associated with improved RV fibrosis, hypertrophy, and RV function. In response to PR, TRN reduced PDGFRβ expression and normalized ERK1/2 activity, which were associated with reduced RV hypertrophy and improved diastolic relaxation. We identify that PDGF drives RV fibroblast proliferation and activation via SMAD2/3, JNK, and β‐catenin signaling. Our studies suggest that TGFβ and PDGF are interconnected drivers of RV fibrosis and hence synergistic targets to improve RV remodeling in RV pressure and volume loading.

Right ventricular (RV) function determines outcomes in RV pressure loading. A better understanding of the time-course and regional distribution of RV remodeling may help optimize targets and timing for therapeutic intervention. We sought to characterize RV remodeling between zero and 6 wk after the initiation of RV pressure loading. Thirty-six rats were randomized to either sham surgery or to pulmonary artery banding (PAB). After echocardiography and conductance catheter studies, groups of rats were euthanized at 1 wk, 3 wk, and 6 wk after sham surgery, or induction of RV pressure loading, for RV histological, RNA, and molecular analysis. A vigorous inflammatory response characterized by increased RV inflammatory cytokines, chemokines, and macrophage markers was observed at 1 wk following PAB. Metabolic changes, transforming growth factor-β (TGF-β)1 canonical signaling, collagenous fibrosis deposition, and apoptosis were already significantly increased by 1 wk after PAB. Genes marking fibroblast activation were upregulated at 1 wk but not at 6 wk post-PAB surgery. Mitochondrial dysfunction was evidenced by increased pyruvate dehydrogenase kinase (PDK) activity and decreased pyruvate dehydrogenase (PDH) phosphorylation significantly at 6-wk post-PAB. These processes preceded the development of overt myocardial hypertrophy and impaired echo parameters of systolic and diastolic function that occurred significantly from 3 wk after PAB. RV myocardial inflammation, metabolic shift, metabolic gene transcription, and profibrotic signaling occur early after initiation of pressure loading when RV pressures are only moderately elevated, before the development of overt myocardial hypertrophy and dysfunction, suggesting that adaptive hypertrophy and maladaptive remodeling occur simultaneously. These results suggest that therapeutic intervention to reduce adverse RV remodeling may be needed earlier and at lower thresholds than currently used.NEW & NOTEWORTHY Exploring the dynamics of right ventricular remodeling: unveiling the intricate interplay between inflammation, metabolic shifts, and fibrotic signaling in response to pressure loading. Through a comprehensive study spanning from initiation to 6 wk post-pressure loading, our research sheds light on the early onset of crucial molecular processes preceding overt hypertrophy and dysfunction. These findings challenge conventional intervention timing, advocating for early, targeted therapeutic strategies to mitigate adverse remodeling in right ventricular pressure loading.

Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbβ3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hinderance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.

Cardiac fibrosis is a key aspect of heart failure, leading to reduced ventricular compliance and impaired electrical conduction in the myocardium. Various pathophysiologic conditions can lead to fibrosis in the left ventricle (LV) and/or right ventricle (RV). Despite growing evidence to support the transcriptomic heterogeneity of cardiac fibroblasts (CFs) in healthy and diseased states, there have been no direct comparisons of CFs in the LV and RV. Given the distinct natures of the ventricles, we hypothesized that LV- and RV-derived CFs would display baseline transcriptomic differences that influence their proliferation and differentiation following injury. Bulk RNA sequencing of CFs isolated from healthy murine left and right ventricles indicated that LV-derived CFs may be further along the myofibroblast transdifferentiation trajectory than cells isolated from the RV. Single-cell RNA-sequencing analysis of the two populations confirmed that Postn+ CFs were more enriched in the LV, whereas Igfbp3+ CFs were enriched in the RV at baseline. Notably, following pressure overload injury, the LV developed a larger subpopulation of pro-fibrotic Thbs4+/Cthrc1+ injury-induced CFs, while the RV showed a unique expansion of two less-well-characterized CF subpopulations (Igfbp3+ and Inmt+). These findings demonstrate that LV- and RV-derived CFs display baseline subpopulation differences that may dictate their diverging responses to pressure overload injury. Further study of these subpopulations will elucidate their role in the development of fibrosis and inform on whether LV and RV fibrosis require distinct treatments.

Introduction: Cigarette smoking (CS) contributes to the most deaths in abdominal aortic aneurysms (AAA) and tobacco use associates with early damage to the abdominal aorta. Associated risk factors, such as hyperlipidemia, link clinical AAA to the atherosclerotic process, suggesting common pathogenetic features with plaque development. We hypothesized that CS exposure exacerbates atherosclerotic disease in the abdominal aorta by increasing the recruitment of inflammatory macrophages that mediate arterial degradation and AAA formation. Methods: Adult male Apoe deficient mice commenced a high cholesterol diet and were concomitantly exposed to CS or room air (RA) for 4, 8, 12, and 16 weeks. As plaque macrophages are highly prevalent in advanced AAA, additional CS exposed mice received a selective CSF1R inhibitor, PLX3397, for up to 16 weeks to deplete macrophages. AAA incidence, atherosclerotic plaque burden and lesion composition were assessed in the aorta by immunofluorescence, Movat, EVG, and Oil Red O staining. Results: CS induced AAA at all timepoints with the highest incidence of 37% at 16 weeks of exposure (n=151 CS, n=75 RA p< 0.05). Aneurysms always coincided with atherosclerosis (n=7 CS, n=7 CS/AAA p<0.05), and severe elastin fragmentation was consistently overlaid by plaque (n=20 CS p<0.05, Fig 1A and B). In some cases, lesions also associated with aortic rupture, causing death in ~11% of animals (n = 35 RA, n = 46 CS p<0.04). CD68+ macrophages associated highly with severe elastin damage but less at intact regions (2.6-fold p<0.0001, Fig 1C). PLX3397-mediated macrophage depletion attenuated plaque development and prevented AAA (n=20 treated, n=27 control p<0.05, Fig 1D). Conclusion: CS exacerbates atherosclerosis and increases the accumulation of macrophages at sites of arterial injury. Depletion of macrophages results in a lack of aneurysm formation, highlighting their capacity to directly injure the aortic wall and necessity to mediate AAA.

Right ventricular (RV) pressure loading induces RV profibrotic signaling and fibrosis associated with RV dysfunction. RV decorin protein levels are decreased in patients with chronic RV pressure loading. RV decorin protein levels are also decreased in 4 animal models of mechanical RV pressure loading and pulmonary arterial hypertension. Human cardiac fibroblasts overexpressing decorin show diminished collagen-1 secretion in response to mechanical or chemical profibrotic stress while decorin knockout human cardiac fibroblasts show increased collagen-1 secretion in response to stress. Downregulation of decorin may play a key role in upregulating transforming growth factor-β1 profibrotic signaling and fibrosis that contribute to RV dysfunction in RV pressure loading.

Cardiac fibrosis and myocardial stiffening are key factors contributing to deteriorating cardiac function during heart failure, especially after myocardial infarction (MI). In the myocardium, cardiac fibroblasts (CFs) are the main cell type driving excess extracellular matrix (ECM) protein deposition, leading to pathological remodelling of the heart. Latent Transforming Growth Factor Binding Protein 2 (LTBP2) is one of the ECM proteins released as part of the fibrotic response and is significantly upregulated in all types of heart failure. Although LTBP2 belongs to a family of proteins that regulate the release of Transforming Growth Factor - β; (TGF-β;) in the ECM, it doesn’t interact with TGF-β;. Nonetheless, LTBP2 is shown to play a role in the progression of fibrosis following heart failure by contributing to CFs activation. Therefore, we hypothesized that in the absence of LTBP2, the heart would have improved cardiac function following MI. We used LTBP2 knockout (KO) C57BL/6 mice and modelled MI by ligating the left anterior descending artery. Echocardiography was performed to analyze the cardiac function in KO and wild-type (WT) infarcted hearts at 7 and 28 days post-MI. KO mice showed significantly better ejection fractions (KO = 24.1±4.9% vs. WT = 9.2±1.4%, p-value < 0.01) and fractional shortening 28 days post-MI compared to WT mice. In conclusion, our findings show that LTBP2 contributes to the deteriorating cardiac function following MI suggesting that targeting LTBP2 may have therapeutic potential. As LTBP2 is known to be a part of the fibrotic response, future studies will investigate the fibrotic profile of WT and KO infarcted hearts using histology and CFs-specific RNA sequencing to highlight the underlying differences leading to the observed cardiac function.

This study reports a new methodology for right heart imaging by ultrasound in mice under right ventricular (RV) pressure over-load. Pulmonary artery constriction (PAC) or sham surgeries were performed on C57BL/6 male mice at 8 wk of age. Ultrasound imaging was conducted at 2, 4, and 8 wk postsurgery using both classical and advanced ultrasound imaging modalities including electrocardiogram (ECG)-based kilohertz visualization, anatomical M-mode, and strain imaging. Based on pulsed Doppler, the PAC group demonstrated dramatically enhanced pressure gradient in the main pulmonary artery (MPA) as compared with the sham group. By the application of advanced imaging modalities in novel short-axis views of the ventricles, the PAC group dem-onstrated increased thickness of RV free wall, enlarged RV chamber, and reduced RV fractional shortening compared with the sham group. The PAC group also showed prolonged RV contraction, asynchronous interplay between RV and left ventricle (LV), and passive leftward motion of the interventricular septum (IVS) at early diastole. Consequently, the PAC group exhibited prolon-gation of LV isovolumic relaxation time, without change in LV wall thickness or systolic function. Significant correlations were found between the maximal pressure gradient in MPA measured by Doppler and the RV systolic pressure by catheterization, as well as the morphological and functional parameters of RV by ultrasound.NEW & NOTEWORTHY The established protocol overcomes the challenges in right heart imaging in mice, thoroughly elucidat-ing the changes of RV, the dynamics of IVS, and the impact on LV and provides new insights into the pathophysiological mecha-nism of RV remodeling.

Cardiac fibrosis is a major risk factor for cardiovascular disease, leading to impaired electrical conduction and reduced ventricular compliance in the heart. Different pathophysiologic conditions can lead to fibrosis in the left ventricle (LV) and/or right ventricle (RV). Despite building evidence to support the transcriptomic heterogeneity of cardiac fibroblasts (CFs) in healthy and diseased states, there have been no direct comparisons of CFs in the LV and RV. Due to the developmental and physiologic differences between the two ventricles, we hypothesized that LV and RV-derived CFs would display transcriptomic differences that influence their proliferation and differentiation following injury. Bulk RNA-seq data from the LVs and RVs of uninjured male mice revealed 442 differentially expressed genes (p<0.05, n=4) with numerous fibrosis-related genes such as Igfbp3 , Col8a1 , Ctgf , Aspn , and Postn being the most significantly different. Single-cell RNA-seq analysis of CFs from uninjured tissue identified nine subpopulations, two of which displayed LV vs RV differences. CFs marked by high expression of Postn , Col8a1 , and Ctgf were more abundant in the LV, whereas CFs marked by high expression of Igfbp3 , Fgl2 , and Sfrp2 were more abundant in the RV. Comparisons with published datasets suggest that Postn -high CFs are primed for differentiation into injury-induced CFs. While the Igfbp3 -high population has not previously been described, top marker genes have mixed pro- and anti-fibrotic roles. We then used surgical models of pressure overload injury (TAC and PAB) to study changes in the transcriptome of LV and RV-derived CFs respectively at 14 days post-surgery. Single cell RNA-seq analysis showed that the LV developed a larger population of pro-fibrotic Thbs4 +/ Cthrc1 + injury-induced CFs while the RV uniquely showed expansion of Igfbp3 - and Inmt -high CFs. Injury experiments were repeated with female mice and showed the same results. These findings demonstrate that LV and RV-derived CFs display subpopulation differences that may cause their diverging responses to pressure overload injury. Further study of these subpopulations will elucidate their role in the development of fibrosis and inform whether LV and RV fibrosis require distinct treatments.

AIMS:Circadian rhythms orchestrate important functions in the cardiovascular system: the contribution of microvascular rhythms to cardiovascular disease progression/severity is unknown. This study hypothesized that (i) myogenic reactivity in skeletal muscle resistance arteries is rhythmic and (ii) disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI).METHODS AND RESULTS:Cremaster skeletal muscle resistance arteries were isolated and assessed using standard pressure myography. Circadian rhythmicity was globally disrupted with the ClockΔ19/Δ19 mutation or discretely through smooth muscle cell-specific Bmal1 deletion (Sm-Bmal1 KO). Cardiac structure and function were determined by echocardiographic, hemodynamic and histological assessments. Myogenic reactivity in cremaster muscle resistance arteries is rhythmic. This rhythm is putatively mediated by the circadian modulation of a mechanosensitive signalosome incorporating tumour necrosis factor and casein kinase 1. Following left anterior descending coronary artery ligation, myogenic responsiveness is locked at the circadian maximum, although circadian molecular clock gene expression cycles normally. Disrupting the molecular clock abolishes myogenic rhythmicity: myogenic tone is suspended at the circadian minimum and is no longer augmented by MI. The reduced myogenic tone in ClockΔ19/Δ19 mice and Sm-Bmal1 KO mice associates with reduced total peripheral resistance (TPR), improved cardiac function and reduced infarct expansion post-MI.CONCLUSIONS:Augmented microvascular constriction aggravates cardiac injury post-MI. Following MI, skeletal muscle resistance artery myogenic reactivity increases specifically within the rest phase, when TPR would normally decline. Disrupting the circadian clock interrupts the MI-induced augmentation in myogenic reactivity: therapeutics targeting the molecular clock, therefore, may be useful for improving MI outcomes.

Heart failure (HF) is associated with pathological remodeling of the myocardium, including the initiation of fibrosis and scar formation by activated cardiac fibroblasts (CFs). Although early CF-dependent scar formation helps prevent cardiac rupture by maintaining the heart's structural integrity, ongoing deposition of the extracellular matrix in the remote and infarct regions can reduce tissue compliance, impair cardiac function, and accelerate progression to HF. In our study, we conducted mass spectrometry (MS) analysis to identify differentially altered proteins and signaling pathways between CFs isolated from 7 day sham and infarcted murine hearts. Surprisingly, CFs from both the remote and infarct regions of injured hearts had a wide number of similarly altered proteins and signaling pathways that were consistent with fibrosis and activation into pathological myofibroblasts. Specifically, proteins enriched in CFs isolated from MI hearts were involved in pathways pertaining to cell-cell and cell-matrix adhesion, chaperone-mediated protein folding, and collagen fibril organization. These results, together with principal component analyses, provided evidence of global CF activation postinjury. Interestingly, however, direct comparisons between CFs from the remote and infarct regions of injured hearts identified 15 differentially expressed proteins between MI remote and MI infarct CFs. Eleven of these proteins (Gpc1, Cthrc1, Vmac, Nexn, Znf185, Sprr1a, Specc1, Emb, Limd2, Pawr, and Mcam) were higher in MI infarct CFs, whereas four proteins (Gstt1, Gstm1, Tceal3, and Inmt) were higher in MI remote CFs. Collectively, our study shows that MI injury induced global changes to the CF proteome, with the magnitude of change reflecting their relative proximity to the site of injury.

A number of diverse G-protein signaling pathways have been shown to regulate insulin secretion from pancreatic β-cells. Accordingly, regulator of G-protein signaling (RGS) proteins have also been implicated in coordinating this process. One such protein, RGS4, is reported to show both positive and negative effects on insulin secretion from β-cells depending on the physiologic context under which it was studied. We here use an RGS4-deficient mouse model to characterize previously unknown G-protein signaling pathways that are regulated by RGS4 during glucose-stimulated insulin secretion from the pancreatic islets. Our data show that loss of RGS4 results in a marked deficiency in glucose-stimulated insulin secretion during both phase I and phase II of insulin release in intact mice and isolated islets. These deficiencies are associated with lower cAMP/PKA activity and a loss of normal calcium surge (phase I) and oscillatory (phase II) kinetics behavior in the RGS4-deficient β-cells, suggesting RGS4 may be important for regulation of both Gαi and Gαq signaling control during glucose-stimulated insulin secretion. Together, these studies add to the known list of G-protein coupled signaling events that are controlled by RGS4 during glucose-stimulated insulin secretion and highlight the importance of maintaining normal levels of RGS4 function in healthy pancreatic tissues.

Arterial stiffening is a significant predictor of cardiovascular disease development and mortality. In elastic arteries, stiffening refers to the loss and fragmentation of elastic fibers, with a progressive increase in collagen fibers. Type VIII collagen (Col-8) is highly expressed developmentally, and then once again dramatically upregulated in aged and diseased vessels characterized by arterial stiffening. Yet its biophysical impact on the vessel wall remains unknown. The purpose of this study was to test the hypothesis that Col-8 functions as a matrix scaffold to maintain vessel integrity during extracellular matrix (ECM) development. These changes are predicted to persist into the adult vasculature, and we have tested this in our investigation. Through our in vivo and in vitro studies, we have determined a novel interaction between Col-8 and elastin. Mice deficient in Col-8 (Col8-/-) had reduced baseline blood pressure and increased arterial compliance, indicating an enhanced Windkessel effect in conducting arteries. Differences in both the ECM composition and VSMC activity resulted in Col8-/- carotid arteries that displayed increased crosslinked elastin and functional distensibility, but enhanced catecholamine-induced VSMC contractility. In vitro studies revealed that the absence of Col-8 dramatically increased tropoelastin mRNA and elastic fiber deposition in the ECM, which was decreased with exogenous Col-8 treatment. These findings suggest a causative role for Col-8 in reducing mRNA levels of tropoelastin and the presence of elastic fibers in the matrix. Moreover, we also found that Col-8 and elastin have opposing effects on VSMC phenotype, the former promoting a synthetic phenotype, whereas the latter confers quiescence. These studies further our understanding of Col-8 function and open a promising new area of investigation related to elastin biology.

Intracellular pools of the heterotrimeric G-protein alpha-subunit, Gαi3, has been shown to promote growth factor signaling, while at the same time inhibiting the activation of JNK and autophagic signaling following nutrient starvation. The precise molecular mechanisms linking Gαi3 to both stress and growth factor signaling remain poorly understood. Importantly, JNK-mediated phosphorylation of Bcl-2 was shown to activate autophagic signaling following nutrient deprivation. Our data shows that activated Gαi3 decreases Bcl-2 phosphorylation, whereas biochemical inhibitors of Gαi3, such as RGS4 and AGS3, markedly increase the levels of phosphorylated Bcl-2. Manipulation of the palmitoylation status and intracellular localization of RGS4 suggests that Gαi3 modulates phosphorylated Bcl-2 levels and autophagic signaling from discreet TGN38-labelled vesicle pools. Consistent with an important role for these molecules in normal tissue responses to nutrient-deprivation, increased Gαi signaling within nutrient-starved adrenal glands from RGS4-KO mice resulted in a dramatic abrogation of autophagic flux, compared to wild type tissues. Together, these data suggest that the activity of Gαi3 and RGS4 from discreet TGN38-labelled vesicle pools are critical regulators of autophagic signaling via their ability to modulate phosphorylation of Bcl-2.