Adeno-associated virus-mediated gene delivery of Perm1 enhances cardiac contractility in mice.

Reduced muscle contractility and mitochondrial bioenergetics are the hallmarks of systolic heart failure. There is currently no therapy targeting both. Here, we show that gene delivery of Perm1 via adeno-associated virus (AAV) simultaneously enhances cardiac contractility and mitochondrial biogenesis in C57BL6 mice. Moreover, we found that PERM1 interacts with troponin C (TnC), a key contractile protein in striated muscle, and that AAV-Perm1 led to the upregulation of TnC. This study suggests that gene delivery of Perm1 may be a novel therapeutic approach to treat systolic heart failure by simultaneously restoring cardiac contractility and mitochondrial bioenergetics.NEW & NOTEWORTHY Perm1 gene delivered with AAV9 enhances cardiac contractility in mice, and it is concomitant with the increase of mitochondrial bioenergetics and upregulation of TnC. This is the first study showing that PERM1, previously known as a striated muscle-specific mitochondrial regulator, also positively regulates cardiac contractility.

Preserving Endothelial Integrity in Human Saphenous Veins during Preparation for Coronary Bypass Surgery.

INTRODUCTION: While multiple factors influence coronary artery bypass graft (CABG) success rates, preserving saphenous vein endothelium during surgery may improve patency. Standard preparations include saphenous vein preparation in heparinized saline (saline) which can result in endothelial loss and damage. Here, we investigated the impact of preparing saphenous graft vessels in heparinized patient blood (blood) versus saline. METHODS: Saphenous vein tissues from a total of 23 patients undergoing CABG were split into 2 groups (1) saline and (2) heparinized patient blood. Excess tissue was fixed for analysis immediately following surgery. Level of endothelial coverage, oxidative stress marker 4-hydroxynonenal (4HNE), and oxidative stress protective marker nuclear factor erythroid 2-related factor 2 (NRF2) were evaluated. RESULTS: In saline patient veins, histological analysis revealed a limited luminal layer, suggesting a loss of endothelial cells (ECs). Immunofluorescent staining of EC markers vascular endothelial cadherin (VE-cadherin) and endothelial nitric oxide identified a significant improvement in EC coverage in the blood versus saline groups. Although both treatment groups expressed 4HNE to similar levels, EC blood samples expressed higher levels of NRF2. CONCLUSION: Our data indicate that use of heparinized patient blood helps preserve the endothelium and promotes vein graft health. This has the potential to improve long-term outcomes in patients.

Connexin 43 across the Vasculature: Gap Junctions and Beyond.

Connexin 43 (Cx43) is essential to the function of the vasculature. Cx43 proteins form gap junctions that allow for the exchange of ions and molecules between vascular cells to facilitate cell-to-cell signaling and coordinate vasomotor activity. Cx43 also has intracellular signaling functions that influence vascular cell proliferation and migration. Cx43 is expressed in all vascular cell types, although its expression and function vary by vessel size and location. This includes expression in vascular smooth muscle cells (vSMC), endothelial cells (EC), and pericytes. Cx43 is thought to coordinate homocellular signaling within EC and vSMC. Cx43 gap junctions also function as conduits between different cell types (heterocellular signaling), between EC and vSMC at the myoendothelial junction, and between pericyte and EC in capillaries. Alterations in Cx43 expression, localization, and post-translational modification have been identified in vascular disease states, including atherosclerosis, hypertension, and diabetes. In this review, we discuss the current understanding of Cx43 localization and function in healthy and diseased blood vessels across all vascular beds.

Mechanisms of Connexin Regulating Peptides.

Gap junctions (GJ) and connexins play integral roles in cellular physiology and have been found to be involved in multiple pathophysiological states from cancer to cardiovascular disease. Studies over the last 60 years have demonstrated the utility of altering GJ signaling pathways in experimental models, which has led to them being attractive targets for therapeutic intervention. A number of different mechanisms have been proposed to regulate GJ signaling, including channel blocking, enhancing channel open state, and disrupting protein-protein interactions. The primary mechanism for this has been through the design of numerous peptides as therapeutics, that are either currently in early development or are in various stages of clinical trials. Despite over 25 years of research into connexin targeting peptides, the overall mechanisms of action are still poorly understood. In this overview, we discuss published connexin targeting peptides, their reported mechanisms of action, and the potential for these molecules in the treatment of disease.

Endothelial-adipocyte Cx43 Mediated Gap Junctions Can Regulate Adiposity.

Obesity is a multifactorial metabolic disorder associated with endothelial dysfunction and increased risk of cardiovascular disease. Adipose capillary adipose endothelial cells (CaECs) plays a crucial role in lipid transport and storage. Here, we investigated the mechanisms underlying CaEC-adipocyte interaction and its impact on metabolic function. Single-cell RNA sequencing (scRNAseq) revealed an enrichment of fatty acid handling machinery in CaECs from high fat diet (HFD) mice, suggesting their specialized role in lipid metabolism. Transmission electron microscopy (TEM) confirmed direct heterocellular contact between CaECs and adipocytes. To model this, we created an in vitro co-culture transwell system to model the heterocellular contact observed with TEM. Contact between ECs and adipocytes in vitro led to upregulation of fatty acid binding protein 4 in response to lipid stimulation, hinting intercellular signaling may be important between ECs and adipocytes. We mined our and others scRNAseq datasets to examine which connexins may be present in adipose capillaries and adipocytes and consistently identified connexin 43 (Cx43) in mouse and humans. Genetic deletion of endothelial Cx43 resulted in increased epididymal fat pad (eWAT) adiposity and dyslipidemia in HFD mice. Consistent with this observation, phosphorylation of Cx43 at serine 368, which closes gap junctions, was increased in HFD mice and lipid-treated ECs. Mice resistant to this post-translational modification, Cx43S368A, were placed on an HFD and were found to have reduced eWAT adiposity and improved lipid profiles. These findings suggest Cx43-mediated heterocellular communication as a possible regulatory mechanism of adipose tissue function.

A Soluble Platelet-Derived Growth Factor Receptor-ß Originates via Pre-mRNA Splicing in the Healthy Brain and is Differentially Regulated during Hypoxia and Aging.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRß) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRß (sPDGFRß) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRß variants, and specifically during tissue homeostasis. Here, we found sPDGFRß protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRß isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRß by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRß transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRß protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRß variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRß variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRß likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRß in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion - critical processes underlying neuronal health and function, and in turn memory and cognition.

A Soluble Platelet-Derived Growth Factor Receptor-ß Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRß) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRß (sPDGFRß) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRß variants, and specifically during tissue homeostasis. Here, we found sPDGFRß protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRß isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRß by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRß transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRß protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRß variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRß variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRß likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRß in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion-critical processes underlying neuronal health and function, and in turn, memory and cognition.

Cell-To-Cell Communication in the Resistance Vasculature.

The arterial vasculature can be divided into large conduit arteries, intermediate contractile arteries, resistance arteries, arterioles, and capillaries. Resistance arteries and arterioles primarily function to control systemic blood pressure. The resistance arteries are composed of a layer of endothelial cells oriented parallel to the direction of blood flow, which are separated by a matrix layer termed the internal elastic lamina from several layers of smooth muscle cells oriented perpendicular to the direction of blood flow. Cells within the vessel walls communicate in a homocellular and heterocellular fashion to govern luminal diameter, arterial resistance, and blood pressure. At rest, potassium currents govern the basal state of endothelial and smooth muscle cells. Multiple stimuli can elicit rises in intracellular calcium levels in either endothelial cells or smooth muscle cells, sourced from intracellular stores such as the endoplasmic reticulum or the extracellular space. In general, activation of endothelial cells results in the production of a vasodilatory signal, usually in the form of nitric oxide or endothelial-derived hyperpolarization. Conversely, activation of smooth muscle cells results in a vasoconstriction response through smooth muscle cell contraction. © 2022 American Physiological Society. Compr Physiol 12: 1-35, 2022.