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The regulation of vascular tone by perivascular tissues is a complex interplay of various paracrine factors. Here, we investigate the anti-contractile effect of skeletal muscle surrounding the femoral and carotid arteries and its underlying mechanisms. Using male and female Wistar rats, we demonstrated that serotonin, phenylephrine, and U-46619 induced a concentration-dependent vasoconstrictor response in femoral artery rings. Interestingly, this response was diminished in the presence of surrounding femoral skeletal muscle, irrespective of sex. No anti-contractile effect was observed when the carotid artery was exposed to its surrounding skeletal muscle. The observed effect in the femoral artery persisted even in the absence of endothelium and when the muscle was detached from the artery. Furthermore, the skeletal muscle surrounding the femoral artery was able to promote an anti-contractile effect in three other vascular beds (basilar, mesenteric, and carotid arteries). Using inhibitors of lactate dehydrogenase and the 1/4 monocarboxylate transporter, we confirmed the involvement of lactate, as both inhibitors were able to abolish the anti-contractile effect. However, lactate did not directly promote vasodilation; rather, it exerted its effect by activating 5' AMP-activated protein kinase (AMPK) and neuronal nitric oxide synthase (NOS1) in the skeletal muscle. Accordingly, Nω-propyl l-arginine, a specific inhibitor of NOS1, prevented the anti-contractile effect, as well as lactate-induced phosphorylation of NOS1 at the stimulatory serine site (1417) in primary skeletal muscle cells. Phosphorylation of NOS1 was reduced in the presence of Bay-3827, a selective AMPK inhibitor. In conclusion, femoral artery-associated skeletal muscle is a potent paracrine and endocrine organ that influences vascular tone in both sexes. Mechanistically, the anti-contractile effect involves muscle fiber type and/or its anatomical location but not the type of artery or its related vascular endothelium. Finally, the femoral artery anti-contractile effect is mediated by the lactate-AMPK-phospho-NOS1Ser1417-NO signaling axis., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)

Modifying surface topography to prevent surface-induced thrombosis in cardiovascular implants allows endothelialization, which is the natural thrombo-resistance of blood-contacting surfaces, and is deemed to be the only long-term solution for hemocompatible materials. We adapted a simulation framework to predict platelet deposition on a modified surface and developed an optimization strategy to promote endothelial retention and limit platelet deposition. Under supraphysiological bulk shear stress, a maximum of 79% linear coverage was achieved. This study concludes that the addition of microtrenches promotes endothelial retention and can be improved through the optimal selection of geometric parameters.

The last few decades have brought contraception to the forefront of research, with great strides made in effectively targeting and optimizing the physiology, pharmacology, and delivery processes that prevent pregnancy. However, these advances still predominantly target female contraceptives for the prevention of contraception, whereas targeting the male sex has lagged far behind. This has led to a marked deficiency in safe and effective male contraceptive agents, resulting in a heavy dependence on female contraceptives to prevent unwanted and unplanned pregnancies. Current research in the veterinary field and in rodents highlights several promising avenues whereby novel, safe, and effective male contraceptive alternatives are being developed-with an emphasis on reduced side effects and reversibility potential. This review aims to discuss current and novel male contraceptives (both human and veterinary formulations) while highlighting their efficacy, advantages, and disadvantages.

In-situ vaccination (ISV) utilizing nanoparticles (NPs) and therapeutic devices like focused ultrasound (FUS) can trigger immune-mediated killing of both treated and untreated cancer cells. However, the impact of confounding factors such as aging and gut microbiota composition on therapeutic outcomes remains poorly understood. In this study, we sequentially treated young mice (∼8 weeks) and old mice (>18 months) with bilateral melanoma using FUS and calreticulin nanoparticles (CRT-NP) to enhance immunogenic cell death. The combination of CRT-NP and FUS (CFUS) demonstrated greater efficacy in inducing regression of treated and untreated tumors in young mice compared to old mice. The diminished effectiveness in older mice was associated with significant differences in gut microbiome composition, characterized by alterations in bacterial species and splenic immune cells. Specifically, young mice exposed to CFUS exhibited higher abundance of Bacteroidetes and Verrucomicrobia, which was not observed in the aged cohorts. Turicibacter, Anaerotruncus, and Ruminiclostridium demonstrated negative correlations with CD8+ T cells but positive correlations with CD4+ T cells and MDSC cells in both age groups. Taxon set enrichment analysis revealed 58 significantly enriched host gene targets in the young cluster compared to only 11 in the aged cluster. These findings highlight the relationship between ISV treatment efficacy and gut microbiome composition, suggesting that interventions such as diet modification, probiotics, or fecal microbiota transplantation may hold potential as therapeutic strategies to enhance immune responses against solid tumors., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

One of the most pronounced changes in the elderly is loss of strength and mobility due to the decline of skeletal muscle function, resulting in a multifactorial condition termed sarcopenia. Although significant clinical changes begin to manifest at advanced ages, recent studies have shown that changes at the cellular and molecular level precede the symptomatology of sarcopenia. By utilizing a single-cell transcriptomic atlas of mouse skeletal muscle across the lifespan, we identified a clear sign of immune senescence that presents during middle age. More importantly, the change in macrophage phenotype in middle age may explain the changes in extracellular matrix composition, especially collagen synthesis, that contributes to fibrosis and overall muscle weakness with advanced age. Our results show a novel paradigm whereby skeletal muscle dysfunction is driven by alterations in tissue-resident macrophages before the appearance of clinical symptoms in middle-aged mice, providing a new therapeutic approach via regulation of immunometabolism.

Clinically serious congenital heart valve defects arise from improper growth and remodeling of endocardial cushions into leaflets. Genetic mutations have been extensively studied but explain less than 20% of cases. Mechanical forces generated by beating hearts drive valve development, but how these forces collectively determine valve growth and remodeling remains incompletely understood. Here, we decouple the influence of those forces on valve size and shape, and study the role of YAP pathway in determining the size and shape. The low oscillatory shear stress promotes YAP nuclear translocation in valvular endothelial cells (VEC), while the high unidirectional shear stress restricts YAP in cytoplasm. The hydrostatic compressive stress activated YAP in valvular interstitial cells (VIC), whereas the tensile stress deactivated YAP. YAP activation by small molecules promoted VIC proliferation and increased valve size. Whereas YAP inhibition enhanced the expression of cell-cell adhesions in VEC and affected valve shape. Finally, left atrial ligation was performed in chick embryonic hearts to manipulate the shear and hydrostatic stress in vivo. The restricted flow in the left ventricle induced a globular and hypoplastic left atrioventricular (AV) valves with an inhibited YAP expression. By contrast, the right AV valves with sustained YAP expression grew and elongated normally. This study establishes a simple yet elegant mechanobiological system by which transduction of local stresses regulates valve growth and remodeling. This system guides leaflets to grow into proper sizes and shapes with the ventricular development, without the need of a genetically prescribed timing mechanism., Competing Interests: MW, BL, SS, CD, FL, JB No competing interests declared, (© 2023, Wang et al.)

Load-bearing soft tissues normally show J-shaped stress-strain behaviors with high compliance at low strains yet high strength at high strains. They have high water content but are still tough and durable. By contrast, naturally derived hydrogels are weak and brittle. Although hydrogels prepared from synthetic polymers can be strong and tough, they do not have the desired bioactivity for emerging biomedical applications. Here, we present a thermomechanical approach to replicate the combinational properties of soft tissues in protein-based photocrosslinkable hydrogels. As a demonstration, we create a gelatin methacryloyl fiber hydrogel with soft tissue-like mechanical properties, such as low Young's modulus (0.1 to 0.3 MPa), high strength (1.1 ± 0.2 MPa), high toughness (9,100 ± 2,200 J/m 3 ), and high fatigue resistance (2,300 ± 500 J/m 2 ). This hydrogel also resembles the biochemical and architectural properties of native extracellular matrix, which enables a fast formation of 3D interconnected cell meshwork inside hydrogels. The fiber architecture also regulates cellular mechanoresponse and supports cell remodeling inside hydrogels. The integration of tissue-like mechanical properties and bioactivity is highly desirable for the next-generation biomaterials and could advance emerging fields such as tissue engineering and regenerative medicine.

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin's inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36-39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36-39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36-39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia., (© 2022. The Author(s).)

Nonalcoholic fatty liver disease (NAFLD) is associated with disruption of homeostatic lipid metabolism, but underlying processes are poorly understood. One possible mechanism is impairment in hepatic circadian rhythm, which regulates key lipogenic mediators in the liver and whose circadian oscillation is diminished in obesity. Nobiletin enhances biological rhythms by activating RAR-related orphan receptor nuclear receptor, protecting against metabolic syndrome in a clock-dependent manner. The effect of nobiletin in NAFLD is unclear. In this study, we investigate the clock-enhancing effects of nobiletin in genetically obese (db/db) PER2::LUCIFERASE reporter mice with fatty liver. We report microarray expression data suggesting hepatic circadian signaling is impaired in db/db mice with profound hepatic steatosis. Circadian PER2 activity, as assessed by mRNA and luciferase assay, was significantly diminished in liver of db/db PER2::LUCIFERASE reporter mice. Continuous animal monitoring systems and constant dark studies suggest the primary circadian defect in db/db mice lies within peripheral hepatic oscillators and not behavioral rhythms or the master clock. In vitro, nobiletin restored PER2 amplitude in lipid-laden PER2::LUCIFERASE reporter macrophages. In vivo, nobiletin dramatically upregulated core clock gene expression, hepatic PER2 activity, and ameliorated steatosis in db/db PER2::LUCIFERASE reporter mice. Mechanistically, nobiletin reduced serum insulin levels, decreased hepatic Srebp1c, Acaca1, Tnfα, and Fgf21 expression, but did not improve Plin2, Plin5, or Cpt1, suggesting nobiletin attenuates steatosis in db/db mice via downregulation of hepatic lipid accumulation. These data suggest restoring endogenous rhythm with nobiletin resolves steatosis in obesity, proposing that hypothesis that targeting the biological clock may be an attractive therapeutic strategy for NAFLD. NEW & NOTEWORTHY NAFLD is the most common chronic liver disease, but underlying mechanisms are unclear. We show here that genetically obese ( db/db ) mice with fatty liver have impaired hepatic circadian rhythm. Hepatic Per2 expression and PER2 reporter activity are diminished in db/db PER2::LUCIFERASE mice. The biological clock-enhancer nobiletin restores hepatic PER2 in db/db PER2::LUCIFERASE mice, resolving steatosis via downregulation of Srebp1c . These studies suggest targeting the circadian clock may be beneficial strategy in NAFLD.

The study of peripheral vasculopathy with chronic metabolic disease is challenged by divergent contributions from spatial (the level of resolution or specific tissue being studied) and temporal origins (evolution of the developing impairments in time). Over many years of studying the development of skeletal muscle vasculopathy and its functional implications, we may be at the point of presenting an integrated conceptual model that addresses these challenges within the obese Zucker rat (OZR) model. At the early stages of metabolic disease, where systemic markers of elevated cardiovascular disease risk are present, the only evidence of vascular dysfunction is at postcapillary and collecting venules, where leukocyte adhesion/rolling is elevated with impaired venular endothelial function. As metabolic disease severity and duration increases, reduced microvessel density becomes evident as well as increased variability in microvascular hematocrit. Subsequently, hemodynamic impairments to distal arteriolar networks emerge, manifesting as increasing perfusion heterogeneity and impaired arteriolar reactivity. This retrograde "wave of dysfunction" continues, creating a condition wherein deficiencies to the distal arteriolar, capillary, and venular microcirculation stabilize and impairments to proximal arteriolar reactivity, wall mechanics, and perfusion distribution evolve. This proximal arteriolar dysfunction parallels increasing failure in fatigue resistance, hyperemic responses, and O 2 uptake within self-perfused skeletal muscle. Taken together, these results present a conceptual model for the retrograde development of peripheral vasculopathy with chronic metabolic disease and provide insight into the timing and targeting of interventional strategies to improve health outcomes. NEW & NOTEWORTHY Working from an established database spanning multiple scales and times, we studied progression of peripheral microvascular dysfunction in chronic metabolic disease. The data implicate the postcapillary venular endothelium as the initiating site for vasculopathy. Indicators of dysfunction, spanning network structures, hemodynamics, vascular reactivity, and perfusion progress in an insidious retrograde manner to present as functional impairments to muscle blood flow and performance much later. The silent vasculopathy progression may provide insight into clinical treatment challenges.

Developmental etiologies causing complex congenital aortic root abnormalities are unknown. Here we show that deletion of Sox17 in aortic root endothelium in mice causes underdeveloped aortic root leading to a bicuspid aortic valve due to the absence of non-coronary leaflet and mispositioned left coronary ostium. The respective defects are associated with reduced proliferation of non-coronary leaflet mesenchyme and aortic root smooth muscle derived from the second heart field cardiomyocytes. Mechanistically, SOX17 occupies a Pdgfb transcriptional enhancer to promote its transcription and Sox17 deletion inhibits the endothelial Pdgfb transcription and PDGFB growth signaling to the non-coronary leaflet mesenchyme. Restoration of PDGFB in aortic root endothelium rescues the non-coronary leaflet and left coronary ostium defects in Sox17 nulls. These data support a SOX17-PDGFB axis underlying aortic root development that is critical for aortic valve and coronary ostium patterning, thereby informing a potential shared disease mechanism for concurrent anomalous aortic valve and coronary arteries., (© 2022. The Author(s).)

Obesity and aging have both seen dramatic increases in prevalence throughout society. This review seeks to highlight common pathologies that present with obesity, along with the underlying risk factors, that have remarkable similarity to what is observed in the aged. These include skeletal muscle dysfunction (loss of quantity and quality), significant increases in adiposity, systemic alterations to autonomic dysfunction, reduction in nitric oxide bioavailability, increases in oxidant stress and inflammation, dysregulation of glucose homeostasis, and mitochondrial dysfunction. This review is organized by the aforementioned indices and succinctly highlights literature that demonstrates similarities between the aged and obese phenotypes in both human and animal models. As aging is an inevitability and obesity prevalence is unlikely to significantly decrease in the near future, these two phenotypes will ultimately combine as a multidimensional syndrome (a pathology termed sarcopenic obesity). Whether the pre-mature aging indices accompanying obesity are additive or synergistic upon entering aging is not yet well defined, but the goal of this review is to illustrate the potential consequences of a double aged phenotype in sarcopenic obesity. Clinically, the modifiable risk factors could be targeted specifically in obesity to allow for increased health span in the aged and sarcopenic obese populations., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Obese individuals are at significantly elevated risk of developing cardiovascular disease (CVD). Additionally, obesity has been associated with disrupted circadian rhythm, manifesting in abnormal sleeping and feeding patterns. To date, the mechanisms linking obesity, circadian disruption, and CVD are incompletely understood, and insight into novel mechanistic pathways is desperately needed to improve therapeutic potential and decrease morbidity and mortality. The objective of this study was to investigate the roles of metabolic and circadian disruptions in obesity and assess their contributions in promoting vascular disease. Lean ( db/+ ) and obese ( db/db ) mice were subjected to 12 weeks of constant darkness to differentiate diurnal and circadian rhythms, and were assessed for changes in metabolism, gene expression, and vascular function. Expression of endothelial nitric oxide synthase (eNOS), an essential enzyme for vascular health, was blunted in obesity and correlated with the oscillatory loss of the novel regulator cezanne (OTUD7B). Lean mice subjected to constant darkness displayed marked reduction in vasodilatory capacity, while endothelial dysfunction of obese mice was not further compounded by diurnal insult. Endothelial gene expression of essential circadian clock components was altered in obesity, but imperfectly phenocopied in lean mice housed in constant darkness, suggesting overlapping but separate mechanisms driving endothelial dysfunction in obesity and circadian disruption. Taken together, these data provide insight into the nature of endothelial circadian rhythm in obesity and suggest a distinct mechanism by which obesity causes a unique circadian defect in the vasculature., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Padgett, Butcher, Haigh, Speese, Corley, Rosewater, Sellers, Larion, Mintz, Fulton and Stepp.)

Cell plasticity plays a key role in embryos by maintaining the differentiation potential of progenitors. Whether postnatal somatic cells revert to an embryonic-like naïve state regaining plasticity and redifferentiate into a cell type leading to a disease remains intriguing. Using genetic lineage tracing and single-cell RNA sequencing, we reveal that Oct4 is induced by nuclear factor κB (NFκB) at embyronic day 9.5 in a subset of mouse endocardial cells originating from the anterior heart forming field at the onset of endocardial-to-mesenchymal transition. These cells acquired a chondro-osteogenic fate. OCT4 in adult valvular aortic cells leads to calcification of mouse and human valves. These calcifying cells originate from the Oct4 embryonic lineage. Genetic deletion of Pou5f1 (Pit-Oct-Unc , OCT4) in the endocardial cell lineage prevents aortic stenosis and calcification of ApoE −/− mouse valve. We established previously unidentified self-cell reprogramming NFκB- and OCT4-mediated inflammatory pathway triggering a dose-dependent mechanism of valve calcification.

Calcific aortic valve disease is dramatically increasing in global burden, yet no therapy exists outside of prosthetic replacement. The increasing proportion of younger and more active patients mandates alternative therapies. Studies suggest a window of opportunity for biologically based diagnostics and therapeutics to alleviate or delay calcific aortic valve disease progression. Advancement, however, has been hampered by limited understanding of the complex mechanisms driving calcific aortic valve disease initiation and progression towards clinically relevant interventions.

Conventional scRNA-seq expression analyses rely on the availability of a high quality genome annotation. Yet, as we show here with scRNA-seq experiments and analyses spanning human, mouse, chicken, mole rat, lemur and sea urchin, genome annotations are often incomplete, in particular for organisms that are not routinely studied. To overcome this hurdle, we created a scRNA-seq analysis routine that recovers biologically relevant transcriptional activity beyond the scope of the best available genome annotation by performing scRNA-seq analysis on any region in the genome for which transcriptional products are detected. Our tool generates a single-cell expression matrix for all transcriptionally active regions (TARs), performs single-cell TAR expression analysis to identify biologically significant TARs, and then annotates TARs using gene homology analysis. This procedure uses single-cell expression analyses as a filter to direct annotation efforts to biologically significant transcripts and thereby uncovers biology to which scRNA-seq would otherwise be in the dark.

Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.

Objective: Calcific aortic valve disease (CAVD) is an actively regulated degenerative disease process. Clinical lesions exhibit marked 3D complexity not represented in current in vitro systems. We here present a unique mechanically stressed 3D culture system that recapitulates valve interstitial cell (VIC) induced matrix calcification through myofibroblastic activation and osteoblastic differentiation. We test the hypothesis that valve endothelial (VEC) - interstitial collaborative interactions modulate the risk and complexity of calcific pathogenesis within mechanically stressed and pro-inflammatory environments., Approach and Results: Porcine aortic valve endothelial and interstitial cells (VEC and VIC) were seeded in a mechanically constrained collagen hydrogels alone or in co-culture configurations. Raised 3D VIC-filled lesions formed within 7 days when cultured in osteogenic media (OGM), and surprisingly exacerbated by endothelial coculture. We identified a spatially coordinated pro-endochondral vs. pro-osteogenic signaling program within the lesion. VEC underwent Endothelial-to-Mesenchymal Transformation (EndMT) and populated the lesion center. The spatial complexity of molecular and cellular signatures of this 3D in vitro CAVD system were consistent with human diseased aortic valve histology. SNAI1 was highly expressed in the VEC and subendothelial direct VIC corroborates with human CAVD lesions. Spatial distribution of Sox9 vs. Runx2 expression within the developed lesions (Sox9 peri-lesion vs. Runx2 predominantly within lesions) mirrored their expression in heavily calcified human aortic valves. Finally, we demonstrate the applicability of this platform for screening potential pharmacologic therapies through blocking the canonical NFκB pathway via BAY 11-7082., Conclusions: Our results establish that VEC actively induce VIC pathological remodeling and calcification via EndMT and paracrine signaling. This mechanically constrained culture platform enables the interrogation of accelerated cell-mediated matrix remodeling behavior underpinned by this cellular feedback circuit. The high fidelity of this complex 3D model system to human CAVD mechanisms supports its use to test mechanisms of intercellular communication in valves and their pharmacological control., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Videographic material of animals can contain inapparent signals, such as color changes or motion that hold information about physiological functions, such as heart and respiration rate, pulse wave velocity, and vocalization. Eulerian video magnification allows the enhancement of such signals to enable their detection. The purpose of this study is to demonstrate how signals relevant to experimental physiology can be extracted from non-contact videographic material of animals., Results: We applied Eulerian video magnification to detect physiological signals in a range of experimental models and in captive and free ranging wildlife. Neotenic Mexican axolotls were studied to demonstrate the extraction of heart rate signal of non-embryonic animals from dedicated videographic material. Heart rate could be acquired both in single and multiple animal setups of leucistic and normally colored animals under different physiological conditions (resting, exercised, or anesthetized) using a wide range of video qualities. Pulse wave velocity could also be measured in the low blood pressure system of the axolotl as well as in the high-pressure system of the human being. Heart rate extraction was also possible from videos of conscious, unconstrained zebrafish and from non-dedicated videographic material of sand lizard and giraffe. This technique also allowed for heart rate detection in embryonic chickens in ovo through the eggshell and in embryonic mice in utero and could be used as a gating signal to acquire two-phase volumetric micro-CT data of the beating embryonic chicken heart. Additionally, Eulerian video magnification was used to demonstrate how vocalization-induced vibrations can be detected in infrasound-producing Asian elephants., Conclusions: Eulerian video magnification provides a technique to extract inapparent temporal signals from videographic material of animals. This can be applied in experimental and comparative physiology where contact-based recordings (e.g., heart rate) cannot be acquired.

A defining characteristic of pulmonary hypertension (PH) is the extensive remodeling of pulmonary arteries (PAs), which results in progressive increases in vascular resistance and stiffness and eventual failure of the right ventricle. There is no cure for PH and identification of novel molecular mechanisms that underlie increased proliferation, reduced apoptosis, and excessive extracellular matrix production in pulmonary artery smooth muscle cells (PASMCs) is a vital objective. Galectin-3 (Gal-3) is a chimeric lectin and potent driver of many aspects of fibrosis, but its role in regulating PASMC behavior in PH remains poorly understood. Herein, we evaluated the importance of increased Gal-3 expression and signaling on PA vascular remodeling and cardiopulmonary function in experimental models of PH. Gal-3 expression was quantified by qRT-PCR, immunoblotting, and immunofluorescence imaging, and its functional role was assessed by specific Gal-3 inhibitors and CRISPR/Cas9-mediated knockout of Gal-3 in the rat. In rat models of PH, we observed increased Gal-3 expression in PASMCs, which stimulated migration and resistance to apoptosis, whereas silencing or genetic deletion reduced cellular migration and PA fibrosis and increased apoptosis. Gal-3 inhibitors attenuated and reversed PA remodeling and fibrosis, as well as hemodynamic indices in monocrotaline (MCT)-treated rats in vivo. These results were supported by genetic deletion of Gal-3 in both MCT and Sugen Hypoxia rat models. In conclusion, our results suggest that elevated Gal-3 levels contribute to inappropriate PA remodeling in PH by enhancing multiple profibrotic mechanisms. Therapeutic strategies targeting Gal-3 may be of benefit in the treatment of PH.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr 198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr 198 -specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr 198 by SRC. We specifically detected SRC-mediated Tyr 198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr 198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr 198 -phosphorylated PANX1 is involved in hypertensive vascular pathology., (© 2019 DeLalio et al.)

Arteriolar endothelial cell-expressed (EC-expressed) α-globin binds endothelial NOS (eNOS) and degrades its enzymatic product, NO, via dioxygenation, thereby lessening the vasodilatory effects of NO on nearby vascular smooth muscle. Although this reaction potentially affects vascular physiology, the mechanisms that regulate α-globin expression and dioxygenase activity in ECs are unknown. Without β-globin, α-globin is unstable and cytotoxic, particularly in its oxidized form, which is generated by dioxygenation and recycled via endogenous reductases. We show that the molecular chaperone α-hemoglobin-stabilizing protein (AHSP) promotes arteriolar α-globin expression in vivo and facilitates its reduction by eNOS. In Ahsp-/- mice, EC α-globin was decreased by 70%. Ahsp-/- and Hba1-/- mice exhibited similar evidence of increased vascular NO signaling, including arteriolar dilation, blunted α1-adrenergic vasoconstriction, and reduced blood pressure. Purified α-globin bound eNOS or AHSP, but not both together. In ECs in culture, eNOS or AHSP enhanced α-globin expression posttranscriptionally. However, only AHSP prevented oxidized α-globin precipitation in solution. Finally, eNOS reduced AHSP-bound α-globin approximately 6-fold faster than did the major erythrocyte hemoglobin reductases (cytochrome B5 reductase plus cytochrome B5). Our data support a model whereby redox-sensitive shuttling of EC α-globin between AHSP and eNOS regulates EC NO degradation and vascular tone.

Cardiac regeneration post-injury is a tantalizing feature of many lower vertebrates such as fishes and urodeles, but absent in adult humans. Restoration of pumping function is a key endpoint of cardiac regeneration, but very little is known about the biomechanical remodeling process. Here, we quantify and compare the evolution of cellular composition and mechanical stiffness of the zebrafish ventricular myocardium during maturation and following cryoinjury during regeneration to better understand the dynamics of biomechanical remodeling during these two processes. With increasing age, normal myocardial trabecular density and cardiomyocyte fraction increased, while non-myocyte cell fractions decreased. Cell density remained constant during maturation. Cardiomyocyte sarcomeres shortened to a minimum reached at 7.5 months of age, but lengthened with additional age. Concomitantly, ventricular wall stiffness increased up until 7.5 months before plateauing with additional age. Endothelial, myofibroblast/smooth muscle, and cardiomyocyte cell fractions were disrupted following cryoinjury, but were progressively restored to age-specific natural norms by 35 days post infarct (DPI). Infarcted myocardium stiffened immediately following cryoinjury and was a 100-fold greater than non-infarcted tissue by 3 DPI. By 14 DPI, stiffness of the infarcted myocardium had fallen below that of 0 DPI and had completely normalized by 35 DPI. Interestingly, cardiomyocyte sarcomere length increased until 14 DPI, but subsequently shortened to lengths below age-specific natural norms, indicating recovery from a volume overloaded condition. These observations are consistent with the view that regenerating myocardium requires biomechanical stimulation (e.g. strain) to rescue from a volume overloaded condition. Intriguingly, the biomechanical progression of the infarcted adult myocardial wall mirrors that of normal remodeling during aging. The biomechanical progression of the infarcted myocardium targets the values of age-specific norms despite a large divergence in initial conditions. These findings identify a novel biomechanical control of heart regeneration that may orchestrate cellular and tissue level remodeling responses.

Growth and remodeling of the primitive pharyngeal arch artery (PAA) network into the extracardiac great vessels is poorly understood but a major source of clinically serious malformations. Undisrupted blood flow is required for normal PAA development, yet specific relationships between hemodynamics and remodeling remain largely unknown. Meeting this challenge is hindered by the common reductionist analysis of morphology to single idealized models, where in fact structural morphology varies substantially. Quantitative technical tools that allow tracking of morphological and hemodynamic changes in a population-based setting are essential to advancing our understanding of morphogenesis. Here, we have developed a methodological pipeline from high-resolution nano-computed tomography imaging and live-imaging flow measurements to multiscale pulsatile computational models. We combine experimental-based computational models of multiple PAAs to quantify hemodynamic forces in the rapidly morphing Hamburger Hamilton (HH) stage HH18, HH24 and HH26 embryos. We identify local morphological variation along the PAAs and their association with specific hemodynamic changes. Population-level mechano-morphogenic variability analysis is a powerful strategy for identifying stage-specific regions of well and poorly tolerated morphological and/or hemodynamic variation that may protect or initiate cardiovascular malformations., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background Obesity compromises cardiometabolic function and is associated with hypertension and chronic kidney disease. Exercise ameliorates these conditions, even without weight loss. Although the mechanisms of exercise's benefits remain unclear, augmented lean body mass is a suspected mechanism. Myostatin is a potent negative regulator of skeletal muscle mass that is upregulated in obesity and downregulated with exercise. The current study tested the hypothesis that deletion of myostatin would increase muscle mass and reduce blood pressure and kidney injury in obesity. Methods and Results Myostatin knockout mice were crossed to db/db mice, and metabolic and cardiovascular functions were examined. Deletion of myostatin increased skeletal muscle mass by ≈50% to 60% without concomitant weight loss or reduction in fat mass. Increased blood pressure in obesity was prevented by the deletion of myostatin, but did not confer additional benefit against salt loading. Kidney injury was evident because of increased albuminuria, which was abolished in obese mice lacking myostatin. Glycosuria, total urine volume, and whole kidney NOX-4 levels were increased in obesity and prevented by myostatin deletion, arguing that increased muscle mass provides a multipronged defense against renal dysfunction in obese mice. Conclusions These experimental observations suggest that loss of muscle mass is a novel risk factor in obesity-derived cardiovascular dysfunction. Interventions that increase muscle mass, either through exercise or pharmacologically, may help limit cardiovascular disease in obese individuals.

Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease., Statement of Significance: We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Ischemic stroke is a leading cause of morbidity and mortality in the US; however, there currently exists only one effective acute pharmacological therapeutic intervention. Purinergic signaling has been shown to regulate vascular function and pathological processes, including inflammation and arterial myogenic reactivity, and plays a role in ischemic stroke outcome. Purinergic signaling requires extracellular purines; however, the mechanism of purine release from cells is unclear. Pannexin1 (Panx1) channels are potentially novel purine release channels expressed throughout the vascular tree that couples regulated purine release with purinergic signaling. Therefore, we examined the role of smooth muscle and endothelial cell Panx1, using conditional cell type-specific transgenic mice, in cerebral ischemia/reperfusion injury outcomes. Deletion of endothelial cell Panx1, but not smooth muscle cell Panx1, significantly reduced cerebral infarct volume after ischemia/reperfusion. Infiltration of leukocytes into brain tissue and development of cerebral myogenic tone were both significantly reduced when mice lacked endothelial Panx1. Panx1 regulation of myogenic tone was unique to the cerebral circulation, as mesenteric myogenic reactivity and blood pressure were independent of endothelial Panx1. Overall, deletion of endothelial Panx1 mitigated cerebral ischemic injury by reducing inflammation and myogenic tone development, indicating that endothelial Panx1 is a possible novel target for therapeutic intervention of ischemic stroke.

Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone., Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone., Methods and Results: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure., Conclusions: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition., (© 2017 American Heart Association, Inc.)

The mitral valve exists in a mechanically demanding environment, with the stress of each cardiac cycle deforming and shearing the native fibroblasts and endothelial cells. Cells and their extracellular matrix exhibit a dynamic reciprocity in the growth and formation of tissue through mechanotransduction and continuously adapt to physical cues in their environment through gene, protein, and cytokine expression. Valve disease is the most common congenital heart defect with watchful waiting and valve replacement surgery the only treatment option. Mitral valve disease (MVD) has been linked to a variety of mechano-active genes ranging from extracellular components, mechanotransductive elements, and cytoplasmic and nuclear transcription factors. Specialized cell receptors, such as adherens junctions, cadherins, integrins, primary cilia, ion channels, caveolae, and the glycocalyx, convert mechanical cues into biochemical responses via a complex of mechanoresponsive elements, shared signaling modalities, and integrated frameworks. Understanding mechanosensing and transduction in mitral valve-specific cells may allow us to discover unique signal transduction pathways between cells and their environment, leading to cell or tissue specific mechanically targeted therapeutics for MVD.

The objective of this study is to test the hypothesis that increased muscle mass has positive effects on cardiovascular function. Specifically, we tested the hypothesis that increases in lean body mass caused by deletion of myostatin improves cardiac performance and vascular function. Echocardiography was used to quantify left ventricular function at baseline and after acute administration of propranolol and isoproterenol to assess β -adrenergic reactivity. Additionally, resistance vessels in several beds were removed, cannulated, pressurized to 60 mmHg and reactivity to vasoactive stimuli was assessed. Hemodynamics were measured using in vivo radiotelemetry. Myostatin deletion results in increased fractional shortening at baseline. Additionally, arterioles in the coronary and muscular microcirculations are more sensitive to endothelial-dependent dilation while nonmuscular beds or the aorta were unaffected. β -adrenergic dilation was increased in both coronary and conduit arteries, suggesting a systemic effect of increased muscle mass on vascular function. Overall hemodynamics and physical characteristics (heart weight and size) remained unchanged. Myostatin deletion mimics in part the effects of exercise on cardiovascular function. It significantly increases lean muscle mass and results in muscle-specific increases in endothelium-dependent vasodilation. This suggests that increases in muscle mass may serve as a buffer against pathological states that specifically target cardiac function (heart failure), the β -adrenergic system (age), and nitric oxide bio-availability (atherosclerosis). Taken together, pharmacological inhibition of the myostatin pathway could prove an excellent mechanism by which the benefits of exercise can be conferred in patients that are unable to exercise., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Polymeric hydrogels have great potential in soft biological micro-actuator applications. However, inappropriate micro-architecture, non-anisotropy, weak biomechanics, and inferior response behaviors limit their development. In this study, we designed and manufactured novel polyacrylonitrile (PAN)-based hydrogel yarns composed with uniaxially aligned nanofibers. The nanofibrous hydrogel yarns possessed anisotropic architecture and robust mechanical properties with flexibility, and could be assembled into defined scaffold structures by subsequent processes. The as-prepared hydrogel yarns showed excellent pH response behaviors, with around 100% maximum length and 900% maximum diameter changes, and the pH response was completed within several seconds. Moreover, the hydrogel yarns displayed unique cell-responsive abilities to promote the cell adhesion, proliferation, and smooth muscle differentiation of human adipose derived mesenchymal stem cells (HADMSC). Chicken cardiomyocytes were further seeded onto our nanofibrous hydrogel yarns to engineer living cell-based microactuators. Our results demonstrated that the uniaxially aligned nanofibrous networks within the hydrogel yarns were the key characteristics leading to the anisotropic organization of cardiac cells, and improved sarcomere organization, mimicking the cardiomyocyte bundles in the native myocardium. The construct is capable of sustaining spontaneous cardiomyocyte pumping behaviors for 7days. Our PAN-based nanofibrous hydrogel yarns are attractive for creating linear microactuators with pH-response capacity and biological microactuators with cardiomyocyte-drivability., Statement of Significance: A mechanically robust polyacrylonitrile-based nanofibrous hydrogel yarn is fabricated by using a modified electrospinning setup in combination with chemical modification processes. The as-prepared hydrogel yarn possesses a uniaxially aligned nanofiber microarchitecture and supports a rapid, pH-dependent expansion/contraction response within a few seconds. Embryonic cardiomyocytes-seeded hydrogel yarn improves the sarcomere organization and mimics the cardiomyocyte bundles in the native myocardium, which sustains spontaneous cardiomyocyte pumping behaviors. The nanofibrous hydrogel yarn has several advantages over traditional bulk hydrogel scaffolds in terms of robust biomechanics, anisotropic aligned architecture, and superior pH response behaviors. Our nanofibrous hydrogel yarn holds the potential to be developed into novel linear and biological microactuators for various biomedical applications., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Atherosclerotic plaque rupture with subsequent embolic events is a major cause of sudden death from myocardial infarction or stroke. Although smooth muscle cells (SMCs) produce and respond to collagens in vitro, there is no direct evidence in vivo that SMCs are a crucial source of collagens and that this impacts lesion development or fibrous cap formation. We sought to determine how conditional SMC-specific knockout of collagen type XV (COL15A1) in SMC lineage tracing mice affects advanced lesion formation given that 1 ) we have previously identified a Col15a1 sequence variant associated with age-related atherosclerosis, 2 ) COL15A1 is a matrix organizer enhancing tissue structural integrity, and 3 ) small interfering RNA-mediated Col15a1 knockdown increased migration and decreased proliferation of cultured human SMCs. We hypothesized that SMC-derived COL15A1 is critical in advanced lesions, specifically in fibrous cap formation. Surprisingly, we demonstrated that SMC-specific Col15a1 knockout mice fed a Western diet for 18 wk failed to form advanced lesions. SMC-specific Col15a1 knockout resulted in lesions reduced in size by 78%, with marked reductions in numbers and proliferating SMCs, and lacked a SMC and extracellular matrix-rich lesion or fibrous cap. In vivo RNA-seq analyses on SMC Col15a1 knockout and wild-type lesions suggested that a mechanism for these effects is through global repression of multiple proatherogenic inflammatory pathways involved in lesion development. These results provide the first direct evidence that a SMC-derived collagen, COL15A1, is critical during lesion pathogenesis, but, contrary to expectations, its loss resulted in marked attenuation rather than exacerbation of lesion pathogenesis. NEW & NOTEWORTHY We report the first direct in vivo evidence that a smooth muscle cell (SMC)-produced collagen, collagen type XV (COL15A1), is critical for atherosclerotic lesion development. SMC Col15a1 knockout markedly attenuated advanced lesion formation, likely through reducing SMC proliferation and impairing multiple proatherogenic inflammatory processes., (Copyright © 2017 the American Physiological Society.)

Regeneration and repair of injured or diseased heart valves remains a clinical challenge. Tissue engineering provides a promising treatment approach to facilitate living heart valve repair and regeneration. Three-dimensional (3D) biomimetic scaffolds that possess heterogeneous and anisotropic features that approximate those of native heart valve tissue are beneficial to the successful in vitro development of tissue engineered heart valves (TEHV). Here we report the development and characterization of a novel composite scaffold consisting of nano- and micro-scale fibrous woven fabrics and 3D hydrogels by using textile techniques combined with bioactive hydrogel formation. Embedded nano-micro fibrous scaffolds within hydrogel enhanced mechanical strength and physical structural anisotropy of the composite scaffold (similar to native aortic valve leaflets) and also reduced its compaction. We determined that the composite scaffolds supported the growth of human aortic valve interstitial cells (HAVIC), balanced the remodeling of heart valve ECM against shrinkage, and maintained better physiological fibroblastic phenotype in both normal and diseased HAVIC over single materials. These fabricated composite scaffolds enable the engineering of a living heart valve graft with improved anisotropic structure and tissue biomechanics important for maintaining valve cell phenotypes., Statement of Significance: Heart valve-related disease is an important clinical problem, with over 300,000 surgical repairs performed annually. Tissue engineering offers a promising strategy for heart valve repair and regeneration. In this study, we developed and tissue engineered living nano-micro fibrous woven fabric/hydrogel composite scaffolds by using textile technique combined with bioactive hydrogel formation. The novelty of our technique is that the composite scaffolds can mimic physical structure anisotropy and the mechanical strength of natural aortic valve leaflet. Moreover, the composite scaffolds prevented the matrix shrinkage, which is major problem that causes the failure of TEHV, and better maintained physiological fibroblastic phenotype in both normal and diseased HAVIC. This work marks the first report of a combination composite scaffold using 3D hydrogel enhanced by nano-micro fibrous woven fabric, and represents a promising tissue engineering strategy to treat heart valve injury., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Aims: Congenital anomalies of arterial valves are common birth defects, leading to valvar stenosis. With no pharmaceutical treatment that can prevent the disease progression, prosthetic replacement is the only choice of treatment, incurring considerable morbidity and mortality. Animal models presenting localized anomalies and stenosis of congenital arterial valves similar to that of humans are critically needed research tools to uncover developmental molecular mechanisms underlying this devastating human condition., Methods and Results: We generated and characterized mouse models with conditionally altered Notch signalling in endothelial or interstitial cells of developing valves. Mice with inactivation of Notch1 signalling in valvar endothelial cells (VEC) developed congenital anomalies of arterial valves including bicuspid aortic valves and valvar stenosis. Notch1 signalling in VEC was required for repressing proliferation and activating apoptosis of valvar interstitial cells (VIC) after endocardial-to-mesenchymal transformation (EMT). We showed that Notch signalling regulated Tnfα expression in vivo, and Tnf signalling was necessary for apoptosis of VIC and post-EMT development of arterial valves. Furthermore, activation or inhibition of Notch signalling in cultured pig aortic VEC-promoted or suppressed apoptosis of VIC, respectively., Conclusion: We have now met the need of critical animal models and shown that Notch-Tnf signalling balances proliferation and apoptosis for post-EMT development of arterial valves. Our results suggest that mutations in its components may lead to congenital anomaly of aortic valves and valvar stenosis in humans., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Background: Ensemble modeling is a promising approach for obtaining robust predictions and coarse grained population behavior in deterministic mathematical models. Ensemble approaches address model uncertainty by using parameter or model families instead of single best-fit parameters or fixed model structures. Parameter ensembles can be selected based upon simulation error, along with other criteria such as diversity or steady-state performance. Simulations using parameter ensembles can estimate confidence intervals on model variables, and robustly constrain model predictions, despite having many poorly constrained parameters., Results: In this software note, we present a multiobjective based technique to estimate parameter or models ensembles, the Pareto Optimal Ensemble Technique in the Julia programming language (JuPOETs). JuPOETs integrates simulated annealing with Pareto optimality to estimate ensembles on or near the optimal tradeoff surface between competing training objectives. We demonstrate JuPOETs on a suite of multiobjective problems, including test functions with parameter bounds and system constraints as well as for the identification of a proof-of-concept biochemical model with four conflicting training objectives. JuPOETs identified optimal or near optimal solutions approximately six-fold faster than a corresponding implementation in Octave for the suite of test functions. For the proof-of-concept biochemical model, JuPOETs produced an ensemble of parameters that gave both the mean of the training data for conflicting data sets, while simultaneously estimating parameter sets that performed well on each of the individual objective functions., Conclusions: JuPOETs is a promising approach for the estimation of parameter and model ensembles using multiobjective optimization. JuPOETs can be adapted to solve many problem types, including mixed binary and continuous variable types, bilevel optimization problems and constrained problems without altering the base algorithm. JuPOETs is open source, available under an MIT license, and can be installed using the Julia package manager from the JuPOETs GitHub repository.

Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer cross-linked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery., Competing Interests: The authors declare no conflict of interest.

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named HbαX) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, HbαX administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II-induced hypertensive states. HbαX acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O 2 binding in red blood cells. In human vasculature, HbαX blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, HbαX modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature., Competing Interests: BEI and LC are co-authors on a patent titled “Composition and methods for modulating arterial tone.” The authors declare no other conflict of interest., (© 2016 American Heart Association, Inc.)

Aims: Valve interstitial cells are active and aggressive players in aortic valve calcification, but their dynamic mediation of mechanically-induced calcific remodeling is not well understood. The goal of this study was to elucidate the feedback loop between valve interstitial cell and calcification mechanics using a novel three-dimensional culture system that allows investigation of the active interplay between cells, disease, and the mechanical valve environment., Methods & Results: We designed and characterized a novel bioreactor system for quantifying aortic valve interstitial cell contractility in 3-D hydrogels in control and osteogenic conditions over 14 days. Interstitial cells demonstrated a marked ability to exert contractile force on their environment and to align collagen fibers with the direction of tension. Osteogenic environment disrupted interstitial cell contractility and led to disorganization of the collagen matrix, concurrent with increased αSMA, TGF-β, Runx2 and calcific nodule formation. Interestingly, RhoA was also increased in osteogenic condition, pointing to an aberrant hyperactivation of valve interstitial cells mechanical activity in disease. This was confirmed by inhibition of RhoA experiments. Inhibition of RhoA concurrent with osteogenic treatment reduced pro-osteogenic signaling and calcific nodule formation. Time-course correlation analysis indicated a significant correlation between interstitial cell remodeling of collagen fibers and calcification events., Conclusions: Interstitial cell contractility mediates internal stress state and organization of the aortic valve extracellular matrix. Osteogenesis disrupts interstitial cell mechanical phenotype and drives disorganization, nodule formation, and pro-calcific signaling via a RhoA-dependent mechanism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Impaired myocardial perfusion reserve (MPR) is prevalent in obesity and diabetes, even in the absence of obstructive coronary artery disease (CAD), and is prognostic of adverse events. We sought to establish the time course of reduced MPR and to investigate associated vascular and tissue properties in mice fed a high-fat diet (HFD), as they are an emerging model of human obesity, diabetes, and reduced MPR without obstructive CAD., Methods: C57Bl/6 mice fed a HFD or a low-fat diet (control) were imaged at 6, 12, 18 and 24 weeks post-diet. The cardiovascular magnetic resonance (CMR) protocol included multi-slice cine imaging to assess ejection fraction (EF), left-ventricular (LV) mass, LV wall thickness (LVWT), and LV volumes, and first-pass perfusion CMR to quantify MPR. Coronary vascular reactivity, aortic atherosclerosis, myocardial capillary density and tissue fibrosis were also assessed., Results: Body weight was increased in HFD mice at 6-24 weeks post-diet (p < 0.05 vs. control). MPR in HFD mice was reduced and LV mass and LVWT were increased in HFD mice at 18 and 24 weeks post-diet (p < 0.05 vs. control). Coronary arteriolar vascular reactivity to adenosine and acetylcholine were reduced in HFD mice (p < 0.05 vs. control). There were no significant differences in cardiac volumes, EF, or capillary density measurements between the two groups. Histology showed interstitial fibrosis in HFD and no aortic atherosclerosis in either group., Conclusions: C57Bl/6 mice fed a HFD for 18-24 weeks have progressively increased LV mass and impaired MPR with fibrosis, normal capillary density and no aortic plaque. These results establish C57Bl/6 mice fed a HFD for 18-24 weeks as a model of impaired MPR without obstructive CAD due to obesity and diabetes.

Objective: Calcific aortic valve (AoV) disease is a significant clinical problem for which the regulatory mechanisms are poorly understood. Enhanced cell-cell adhesion is a common mechanism of cellular aggregation, but its role in calcific lesion formation is not known. Cadherin-11 (Cad-11) has been associated with lesion formation in vitro, but its function during adult valve homeostasis and pathogenesis is not known. This study aims to elucidate the specific functions of Cad-11 and its downstream targets, RhoA and Sox9, in extracellular matrix remodeling and AoV calcification., Approach and Results: We conditionally overexpressed Cad-11 in murine heart valves using a novel double-transgenic Nfatc1(Cre);R26-Cad11(TglTg) mouse model. These mice developed hemodynamically significant aortic stenosis with prominent calcific lesions in the AoV leaflets. Cad-11 overexpression upregulated downstream targets, RhoA and Sox9, in the valve interstitial cells, causing calcification and extensive pathogenic extracellular matrix remodeling. AoV interstitial cells overexpressing Cad-11 in an osteogenic environment in vitro rapidly form calcific nodules analogous to in vivo lesions. Molecular analyses revealed upregulation of osteoblastic and myofibroblastic markers. Treatment with a Rho-associated protein kinase inhibitor attenuated nodule formation, further supporting that Cad-11-driven calcification acts through the small GTPase RhoA/Rho-associated protein kinase signaling pathway., Conclusions: This study identifies one of the underlying molecular mechanisms of heart valve calcification and demonstrates that overexpression of Cad-11 upregulates RhoA and Sox9 to induce calcification and extracellular matrix remodeling in adult AoV pathogenesis. The findings provide a potential molecular target for clinical treatment., (© 2016 American Heart Association, Inc.)

Unlabelled: Calcific aortic valve disease (CAVD) progression is a highly dynamic process whereby normally fibroblastic valve interstitial cells (VIC) undergo osteogenic differentiation, maladaptive extracellular matrix (ECM) composition, structural remodeling, and tissue matrix stiffening. However, how VIC with different phenotypes dynamically affect matrix properties and how the altered matrix further affects VIC phenotypes in response to physiological and pathological conditions have not yet been determined. In this study, we develop 3D hydrogels with tunable matrix stiffness to investigate the dynamic interplay between VIC phenotypes and matrix biomechanics. We find that VIC populated within hydrogels with valve leaflet like stiffness differentiate towards myofibroblasts in osteogenic media, but surprisingly undergo osteogenic differentiation when cultured within lower initial stiffness hydrogels. VIC differentiation progressively stiffens the hydrogel microenvironment, which further upregulates both early and late osteogenic markers. These findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive RhoA/ROCK signaling pathway, delays the osteogenic differentiation process. Therefore, direct ECM biomechanical modulation can affect VIC phenotypes towards and against osteogenic differentiation in 3D culture. These findings highlight the importance of the homeostatic maintenance of matrix stiffness to restrict pathological VIC differentiation., Statement of Significance: We implement 3D hydrogels with tunable matrix stiffness to investigate the dynamic interaction between valve interstitial cells (VIC, major cell population in heart valve) and matrix biomechanics. This work focuses on how human VIC responses to changing 3D culture environments. Our findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification, which is the hallmark of calcific aortic valve disease. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive signaling pathway, delays VIC osteogenic differentiation. Our findings provide an improved understanding of VIC-matrix interactions to aid in interpretation of VIC calcification studies in vitro and suggest that ECM disruption resulting in local tissue stiffness decreases may promote calcific aortic valve disease., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A major challenge facing public health is the increased incidence and prevalence of the metabolic syndrome, a clinical condition characterized by excess adiposity, impaired glycaemic control, dyslipidaemia and moderate hypertension. The greatest concern for this syndrome is the profound increase in risk for development of peripheral vascular disease (PVD) in afflicted persons. However, ongoing studies suggest that reductions in bulk blood flow to skeletal muscle may not be the primary contributor to the premature muscle fatigue that is a hallmark of PVD. Compelling evidence has been provided suggesting that an increasingly spatially heterogeneous and temporally stable distribution of blood flow at successive arteriolar bifurcations in metabolic syndrome creates an environment where a large number of the pre-capillary arterioles have low perfusion, low haematocrit, and are increasingly confined to this state, with limited ability to adapt perfusion in response to a challenged environment. Single pharmacological interventions are unable to significantly restore function owing to a divergence in their spatial effectiveness, although combined therapeutic approaches to correct adrenergic dysfunction, elevated oxidant stress and increased thromboxane A2 improve perfusion-based outcomes. Integrated, multi-target therapeutic interventions designed to restore healthy network function and flexibility may provide for superior outcomes in subjects with metabolic syndrome-associated PVD., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

To determine the impact of progressive elevations in peripheral vascular disease (PVD) risk on microvascular function, we utilized eight rat models spanning "healthy" to "high PVD risk" and used a multiscale approach to interrogate microvascular function and outcomes: healthy: Sprague-Dawley rats (SDR) and lean Zucker rats (LZR); mild risk: SDR on high-salt diet (HSD) and SDR on high-fructose diet (HFD); moderate risk: reduced renal mass-hypertensive rats (RRM) and spontaneously hypertensive rats (SHR); high risk: obese Zucker rats (OZR) and Dahl salt-sensitive rats (DSS). Vascular reactivity and biochemical analyses demonstrated that even mild elevations in PVD risk severely attenuated nitric oxide (NO) bioavailability and caused progressive shifts in arachidonic acid metabolism, increasing thromboxane A2 levels. With the introduction of hypertension, arteriolar myogenic activation and adrenergic constriction were increased. However, while functional hyperemia and fatigue resistance of in situ skeletal muscle were not impacted with mild or moderate PVD risk, blood oxygen handling suggested an increasingly heterogeneous perfusion within resting and contracting skeletal muscle. Analysis of in situ networks demonstrated an increasingly stable and heterogeneous distribution of perfusion at arteriolar bifurcations with elevated PVD risk, a phenomenon that was manifested first in the distal microcirculation and evolved proximally with increasing risk. The increased perfusion distribution heterogeneity and loss of flexibility throughout the microvascular network, the result of the combined effects on NO bioavailability, arachidonic acid metabolism, myogenic activation, and adrenergic constriction, may represent the most accurate predictor of the skeletal muscle microvasculopathy and poor health outcomes associated with chronic elevations in PVD risk., (Copyright © 2016 the American Physiological Society.)

During valvulogenesis, globular endocardial cushions elongate and remodel into highly organized thin fibrous leaflets. Proper regulation of this dynamic process is essential to maintain unidirectional blood flow as the embryonic heart matures. In this study, we tested how mechanosensitive small GTPases, RhoA and Rac1, coordinate atrioventricular valve (AV) differentiation and morphogenesis. RhoA activity and its regulated GTPase-activating protein FilGAP are elevated during early cushion formation but decreased considerably during valve remodeling. In contrast, Rac1 activity was nearly absent in the early cushions but increased substantially as the valve matured. Using gain- and loss-of-function assays, we determined that the RhoA pathway was essential for the contractile myofibroblastic phenotype present in early cushion formation but was surprisingly insufficient to drive matrix compaction during valve maturation. The Rac1 pathway was necessary to induce matrix compaction in vitro through increased cell adhesion, elongation, and stress fiber alignment. Facilitating this process, we found that acute cyclic stretch was a potent activator of RhoA and subsequently downregulated Rac1 activity via FilGAP. On the other hand, chronic cyclic stretch reduced active RhoA and downstream FilGAP, which enabled Rac1 activation. Finally, we used partial atrial ligation experiments to confirm in vivo that altered cyclic mechanical loading augmented or restricted cushion elongation and thinning, directly through potentiation of active Rac1 and active RhoA, respectively. Together, these results demonstrate that cyclic mechanical signaling coordinates the RhoA to Rac1 signaling transition essential for proper embryonic mitral valve remodeling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

Proper remodeling of the endocardial cushions into thin fibrous valves is essential for gestational progression and long-term function. This process involves dynamic interactions between resident cells and their local environment, much of which is not understood. In this study, we show that deficiency of the cell-cell adhesion protein cadherin-11 (Cad-11) results in significant embryonic and perinatal lethality primarily due to valve related cardiac dysfunction. While endocardial to mesenchymal transformation is not abrogated, mesenchymal cells do not homogeneously cellularize the cushions. These cushions remain thickened with disorganized ECM, resulting in pronounced aortic valve insufficiency. Mice that survive to adulthood maintain thickened and stenotic semilunar valves, but interestingly do not develop calcification. Cad-11 (-/-) aortic valve leaflets contained reduced Sox9 activity, β1 integrin expression, and RhoA-GTP activity, suggesting that remodeling defects are due to improper migration and/or cellular contraction. Cad-11 deletion or siRNA knockdown reduced migration, eliminated collective migration, and impaired 3D matrix compaction by aortic valve interstitial cells (VIC). Cad-11 depleted cells in culture contained few filopodia, stress fibers, or contact inhibited locomotion. Transfection of Cad-11 depleted cells with constitutively active RhoA restored cell phenotypes. Together, these results identify cadherin-11 mediated adhesive signaling for proper remodeling of the embryonic semilunar valves., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Inflammatory cell recruitment to local sites of tissue injury and/or infection is controlled by a plethora of signalling processes influencing cell-to-cell interactions between the vascular endothelial cells (ECs) in post-capillary venules and circulating leukocytes. Recently, ATP-sensitive P2Y purinergic receptors have emerged as downstream regulators of EC activation in vascular inflammation. However, the mechanism(s) regulating cellular ATP release in this response remains elusive. Here we report that the ATP-release channel Pannexin1 (Panx1) opens downstream of EC activation by TNF-α. This process involves activation of type-1 TNF receptors, recruitment of Src family kinases (SFK) and SFK-dependent phosphorylation of Panx1. Using an inducible, EC-specific Panx1 knockout mouse line, we report a previously unidentified role for Panx1 channels in promoting leukocyte adhesion and emigration through the venous wall during acute systemic inflammation, placing Panx1 channels at the centre of cytokine crosstalk with purinergic signalling in the endothelium.

MYBPC3 mutations cause hypertrophic cardiomyopathy, which is frequently associated with mitral valve (MV) pathology. We reasoned that increased MV size is caused by localized growth factors with paracrine effects. We used high-resolution echocardiography to compare Mybpc3 -null, heterozygous, and wild-type mice ( n = 84, aged 3-6 months) and micro-CT for MV volume ( n = 6, age 6 months). Mybpc3 -null mice showed left ventricular hypertrophy, dilation, and systolic dysfunction compared to heterozygous and wild-type mice, but no systolic anterior motion of the MV or left ventricular outflow obstruction. Compared to wild-type mice, echocardiographic anterior leaflet length (adjusted for left ventricular size) was greatest in Mybpc3 -null mice (1.92 ± 0.08 vs . 1.72 ± 0.08 mm, p < 0.001), as was combined leaflet thickness (0.23 ± 0.04 vs . 0.15 ± 0.02 mm, p < 0.001). Micro-CT analyses of Mybpc3 -null mice demonstrated increased MV volume (0.47 ± 0.06 vs . 0.15 ± 0.06 mm 3 , p = 0.018) and thickness (0.35 ± 0.04 vs . 0.12 ± 0.04 mm, p = 0.002), coincident with increased markers of TGFβ activity compared to heterozygous and wild-type littermates. Similarly, excised MV from a patient with MYBPC3 mutation showed increased TGFβ activity. We conclude that MYBPC3 deficiency causes hypertrophic cardiomyopathy with increased MV leaflet length and thickness despite the absence of left ventricular outflow-tract obstruction, in parallel with increased TGFβ activity. MV changes in hypertrophic cardiomyopathy may be due to paracrine effects, which represent targets for therapeutic studies.

Evolution of metabolic syndrome is associated with a progressive reduction in skeletal muscle microvessel density, known as rarefaction. Although contributing to impairments to mass transport and exchange, the temporal development of rarefaction and the contributing mechanisms that lead to microvessel loss are both unclear and critical areas for investigation. Although previous work suggests that rarefaction severity in obese Zucker rats (OZR) is predicted by the chronic loss of vascular nitric oxide (NO) bioavailability, we have determined that this hides a biphasic development of rarefaction, with both early and late components. Although the total extent of rarefaction was well predicted by the loss in NO bioavailability, the early pulse of rarefaction developed before a loss of NO bioavailability and was associated with altered venular function (increased leukocyte adhesion/rolling), and early elevation in oxidant stress, TNF-α levels, and the vascular production of thromboxane A2 (TxA2). Chronic inhibition of TNF-α blunted the severity of rarefaction and also reduced vascular oxidant stress and TxA2 production. Chronic blockade of the actions of TxA2 also blunted rarefaction, but did not impact oxidant stress or inflammation, suggesting that TxA2 is a downstream outcome of elevated reactive oxygen species and inflammation. If chronic blockade of TxA2 is terminated, microvascular rarefaction in OZR skeletal muscle resumes, but at a reduced rate despite low NO bioavailability. These results suggest that therapeutic interventions against inflammation and TxA2 under conditions where metabolic syndrome severity is moderate or mild may prevent the development of a condition of accelerated microvessel loss with metabolic syndrome., (Copyright © 2014 the American Physiological Society.)