Pyroptosis in cardiovascular diseases: Pumping gasdermin on the fire.

Pyroptosis is a form of programmed cell death associated with activation of inflammasomes and inflammatory caspases, proteolytic cleavage of gasdermin proteins (forming pores in the plasma membrane), and selective release of proinflammatory mediators. Induction of pyroptosis results in amplification of inflammation, contributing to the pathogenesis of chronic cardiovascular diseases such as atherosclerosis and diabetic cardiomyopathy, and acute cardiovascular events, such as thrombosis and myocardial infarction. While engagement of pyroptosis during sepsis-induced cardiomyopathy and septic shock is expected and well documented, we are just beginning to understand pyroptosis involvement in the pathogenesis of cardiovascular diseases with less defined inflammatory components, such as atrial fibrillation. Due to the danger that pyroptosis represents to cells within the cardiovascular system and the whole organism, multiple levels of pyroptosis regulation have evolved. Those include regulation of inflammasome priming, post-translational modifications of gasdermins, and cellular mechanisms for pore removal. While pyroptosis in macrophages is well characterized as a dramatic pro-inflammatory process, pyroptosis in other cell types within the cardiovascular system displays variable pathways and consequences. Furthermore, different cells and organs engage in local and distant crosstalk and exchange of pyroptosis triggers (oxidized mitochondrial DNA), mediators (IL-1ß, S100A8/A9) and antagonists (IL-9). Development of genetic tools, such as Gasdermin D knockout animals, and small molecule inhibitors of pyroptosis will not only help us fully understand the role of pyroptosis in cardiovascular diseases but may result in novel therapeutic approaches inhibiting inflammation and progression of chronic cardiovascular diseases to reduce morbidity and mortality from acute cardiovascular events.

The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation.

Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.

Unfolded Protein Response Differentially Modulates the Platelet Phenotype.

BACKGROUND: Unfolded protein response (UPR) is a multifaceted signaling cascade that alleviates protein misfolding. Although well studied in nucleated cells, UPR in absence of transcriptional regulation has not been described. Intricately associated with cardiovascular diseases, platelets, despite being anucleate, respond rapidly to stressors in blood. We investigate the UPR in anucleate platelets and explore its role, if any, on platelet physiology and function. METHODS: Human and mouse platelets were studied using a combination of ex vivo and in vivo experiments. Platelet lineage-specific knockout mice were generated independently for each of the 3 UPR pathways, PERK (protein kinase RNA [PKR]-like endoplasmic reticulum kinase), XBP1 (X-binding protein), and ATF6 (activating transcription factor 6). Diabetes patients were prospectively recruited, and platelets were evaluated for activation of UPR under chronic pathophysiological disease conditions. RESULTS: Tunicamycin induced the IRE1α (inositol-requiring enzyme-1alpha)-XBP1 pathway in human and mouse platelets, while oxidative stress predominantly activated the PERK pathway. PERK deletion significantly increased platelet aggregation and apoptosis and phosphorylation of PLCγ2, PLCß3, and p38 MAPK. Deficiency of XBP1 increased platelet aggregation, with higher PLCß3 and PKCδ activation. ATF6 deletion mediated a relatively modest effect on platelet phenotype with increased PKA (protein kinase A). Platelets from diabetes patients exhibited a positive correlation between disease severity, platelet activation, and protein aggregation, with only IRE1α-XBP1 activation. Moreover, IRE1α inhibition increased platelet aggregation, while clinically approved chemical chaperone, sodium 4-phenylbutyrate reduced the platelet hyperactivation. CONCLUSIONS: We show for the first time, that UPR activation occurs in platelets and can be independent of genomic regulation, with selective induction being specific to the source and severity of stress. Each UPR pathway plays a key role and can differentially modulate the platelet activation pathways and phenotype. Targeting the specific arms of UPR may provide a new antiplatelet strategy to mitigate thrombotic risk in diabetes and other cardiovascular diseases.

Novel strategies for targeting neutrophil against myocardial infarction.

Inflammation is a crucial factor in cardiac remodeling after acute myocardial infarction (MI). Neutrophils, as the first wave of leukocytes to infiltrate the injured myocardium, exacerbate inflammation and cardiac injury. However, therapies that deplete neutrophils to manage cardiac remodeling after MI have not consistently produced promising outcomes. Recent studies have revealed that neutrophils at different time points and locations may have distinct functions. Thus, transferring neutrophil phenotypes, rather than simply blocking their activities, potentially meet the needs of cardiac repair. In this review, we focus on discussing the fate, heterogeneity, functions of neutrophils, and attempt to provide a more comprehensive understanding of their roles and targeting strategies in MI. We highlight the strategies and translational potential of targeting neutrophils to limit cardiac injury to reduce morbidity and mortality from MI.

Histone Acetyltransferases p300 and CBP Coordinate Distinct Chromatin Remodeling Programs in Vascular Smooth Muscle Plasticity.

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. METHODS: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300iKO or CBPiKO), and in samples of human intimal hyperplasia. RESULTS: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBPiKO mice were entirely protected. In normal aorta, p300iKO reduced, but CBPiKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. CONCLUSIONS: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. METHODS: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. RESULTS: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. CONCLUSIONS: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

TET2 Protects Against Vascular Smooth Muscle Cell Apoptosis and Intimal Thickening in Transplant Vasculopathy.

BACKGROUND: Coronary allograft vasculopathy (CAV) is a devastating sequela of heart transplant in which arterial intimal thickening limits coronary blood flow. There are currently no targeted therapies to prevent or reduce this pathology that leads to transplant failure. Vascular smooth muscle cell (VSMC) phenotypic plasticity is critical in CAV neointima formation. TET2 (TET methylcytosine dioxygenase 2) is an important epigenetic regulator of VSMC phenotype, but the role of TET2 in the progression of CAV is unknown. METHODS: We assessed TET2 expression and activity in human CAV and renal transplant samples. We also used the sex-mismatched murine aortic graft model of graft arteriopathy (GA) in wild-type and inducible smooth muscle-specific Tet2 knockout mice; and in vitro studies in murine and human VSMCs using knockdown, overexpression, and transcriptomic approaches to assess the role of TET2 in VSMC responses to IFNγ (interferon γ), a cytokine elaborated by T cells that drives CAV progression. RESULTS: In the present study, we found that TET2 expression and activity are negatively regulated in human CAV and renal transplant samples and in the murine aortic graft model of GA. IFNγ was sufficient to repress TET2 and induce an activated VSMC phenotype in vitro. TET2 depletion mimicked the effects of IFNγ, and TET2 overexpression rescued IFNγ-induced dedifferentiation. VSMC-specific TET2 depletion in aortic grafts, and in the femoral wire restenosis model, resulted in increased VSMC apoptosis and medial thinning. In GA, this apoptosis was tightly correlated with proliferation. In vitro, TET2-deficient VSMCs undergo apoptosis more readily in response to IFNγ and expressed a signature of increased susceptibility to extrinsic apoptotic signaling. Enhancing TET2 enzymatic activity with high-dose ascorbic acid rescued the effect of GA-induced VSMC apoptosis and intimal thickening in a TET2-dependent manner. CONCLUSIONS: TET2 is repressed in CAV and GA, likely mediated by IFNγ. TET2 serves to protect VSMCs from apoptosis in the context of transplant vasculopathy or IFNγ stimulation. Promoting TET2 activity in vivo with systemic ascorbic acid reduces VSMC apoptosis and intimal thickening. These data suggest that promoting TET2 activity in CAV may be an effective strategy for limiting CAV progression.

Circular RNA CircMAP3K5 Acts as a MicroRNA-22-3p Sponge to Promote Resolution of Intimal Hyperplasia Via TET2-Mediated Smooth Muscle Cell Differentiation.

BACKGROUND: Aberrant expression of circular RNA contributes to human diseases. Circular RNAs regulate gene expression by sequestering specific microRNAs. In this study, we investigated whether circMAP3K5 (circular mitogen-activated protein kinase 5) could act as a competing endogenous microRNA-22-3p (miR-22-3p) sponge and regulate neointimal hyperplasia. METHODS: Circular RNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (SMCs) treated with or without platelet-derived growth factor. Expression levels of circMAP3K5 were assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy or coronary heart disease. The role of circMAP3K5 in intimal hyperplasia was further investigated in mice with adeno-associated virus 9-mediated circMAP3K5 transfection. SMC-specific Tet2 (ten-eleven translocation-2) knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which circMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. RESULTS: RNA sequencing demonstrated that treatment with platelet-derived growth factor-BB significantly reduced expression of circMAP3K5 in human coronary artery SMCs. Wire-injured mouse femoral arteries and diseased arteries from patients with coronary heart disease (where platelet-derived growth factor-BB is increased) confirmed in vivo downregulation of circMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of circMAP3K5 inhibited the proliferation of human coronary artery SMCs. In vivo adeno-associated virus 9-mediated transfection of circMap3k5 (mouse circular Map3k5) specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, circMAP3K5 (human circular MAP3K5) was found to sequester miR-22-3p, which, in turn, inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the antiproliferative effect of circMap3k5 on vascular SMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the circMap3k5-mediated antiproliferative effect on vascular SMCs. CONCLUSIONS: We identify circMAP3K5 as a master regulator of TET2-mediated vascular SMC differentiation. Targeting the circMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia, including restenosis and atherosclerosis.

Bone marrow-derived naïve B lymphocytes improve heart function after myocardial infarction: a novel cardioprotective mechanism for empagliflozin.

The role of adaptive immunity in myocardial recovery post myocardial infarction (MI), particularly the immune response by B lymphocytes, remains elusive. Bone marrow immune microenvironment in response to MI is remotely regulated by the hypothalamic pituitary adrenal (HPA) axis. We utilized the cardioprotective actions of SGLT2 inhibitor to identify and characterize bone marrow B cell subsets that respond to myocardial injury. Initially, we preformed ligation of left anterior descendant (LAD) coronary artery in male C57BL/6J mice to monitor the dynamic changes of immune cells across tissues. Mechanistic insights from mouse models demonstrated arrest of bone marrow B cell maturation and function 24 h post MI. A secondary MI model (twice MIs) in mice was established for the first time to evaluate the dosage-dependent cardioprotection of empagliflozin (EMPA). Single-cell RNA-Seq further demonstrated that EMPA restored bone marrow naïve B cell (B220+CD19+CD43-IgM+IgD+) counts and function. Additionally, we recruited 14 acute MI patients with single LAD disease, and profiled B cells post percutaneous coronary intervention (PCI) (compared to 18 matched no-MI controls). We revealed a positive correlation of increased B cell counts with enhanced ejection fraction in MI patients with PCI while lymphopenia was associated with patients with heart failure. Mechanistically, MI triggers the release of glucocorticoids from neuroendocrine system, inducing NHE1-mediated autophagic death of bone marrow B cells while repressing B cell progenitor proliferation and differentiation. Infusion of B cells derived from bone marrow significantly improved cardiac function and diminished infarct size post MI. These findings provide new mechanistic insights into regulation of adaptive immune response post MI, and support targeting bone marrow B cell development for improved ventricular remodeling and reduced heart failure after MI.

Reduced Platelet miR-223 Induction in Kawasaki Disease Leads to Severe Coronary Artery Pathology Through a miR-223/PDGFRß Vascular Smooth Muscle Cell Axis.

RATIONALE: Kawasaki disease (KD) is an acute vasculitis of early childhood that can result in permanent coronary artery structural damage. The cause for this arterial vulnerability in up to 15% of patients with KD is unknown. Vascular smooth muscle cell dedifferentiation play a key role in the pathophysiology of medial damage and aneurysm formation, recognized arterial pathology in KD. Platelet hyperreactivity is also a hallmark of KD. We recently demonstrated that uptake of platelets and platelet-derived miRNAs influences vascular smooth muscle cell phenotype in vivo. OBJECTIVE: We set out to explore whether platelet/vascular smooth muscle cell (VSMC) interactions contribute to coronary pathology in KD. METHODS AND RESULTS: We prospectively recruited and studied 242 patients with KD, 75 of whom had documented coronary artery pathology. Genome-wide miRNA sequencing and droplet digital PCR demonstrated that patient with KD platelets have significant induction of miR-223 compared with healthy controls (HCs). Platelet-derived miR-223 has recently been shown to promote vascular smooth muscle quiescence and resolution of wound healing after vessel injury. Paradoxically, patients with KD with the most severe coronary pathology (giant coronary artery aneurysms) exhibited a lack of miR-223 induction. Hyperactive platelets isolated from patients with KD are readily taken up by VSMCs, delivering functional miR-223 into the VSMCs promoting VSMC differentiation via downregulation of PDGFRß (platelet-derived growth factor receptor ß). The lack of miR-223 induction in patients with severe coronary pathology leads to persistent VSMC dedifferentiation. In a mouse model of KD (Lactobacillus casei cell wall extract injection), miR-223 knockout mice exhibited increased medial thickening, loss of contractile VSMCs in the media, and fragmentation of medial elastic fibers compared with WT mice, which demonstrated significant miR-223 induction upon Lactobacillus casei cell wall extract challenge. The excessive arterial damage in the miR-223 knockout could be rescued by adoptive transfer of platelet, administration of miR-223 mimics, or the PDGFRß inhibitor imatinib mesylate. Interestingly, miR-223 levels progressively increase with age, with the lowest levels found in <5-year-old. This provides a basis for coronary pathology susceptibility in this very young cohort. CONCLUSIONS: Platelet-derived miR-223 (through PDGFRß inhibition) promotes VSMC differentiation and resolution of KD induced vascular injury. Lack of miR-223 induction leads to severe coronary pathology characterized by VSMC dedifferentiation and medial damage. Detection of platelet-derived miR-223 in patients with KD (at the time of diagnosis) may identify patients at greatest risk of coronary artery pathology. Moreover, targeting platelet miR-223 or VSMC PDGFRß represents potential therapeutic strategies to alleviate coronary pathology in KD. Graphic Abstract: A graphic abstract is available for this article.

Association of iron infusion reactions with ABO blood type.

OBJECTIVES: We sought to determine risk factors for iv iron infusion-related reactions (IRR), and identify strategies for iron repletion after IRR. METHODS: We conducted a retrospective chart review of patients treated in the classical hematology clinic at Yale Cancer Center (n = 330 consecutive patients) from 2016 to 2021, who received iv ferumoxytol (60.3%), iron sucrose (14.8%), or iron dextran (10.9%). RESULTS: The iv iron IRR was noted in 58 (17.6%) patients, 62.1% of whom had previously tolerated iv iron. The severity of IRR was mild in 22, moderate in 23, and severe in 11 patients. Most (72.4%) patients who experienced IRR tolerated a subsequent iv iron infusion. On multivariable analysis, a history of non-medication allergies was associated with greater odds of IRR (odds ratio [OR] 2.12, 95% confidence interval (CI): 1.16-3.87, p = .01). No patients with type AB blood, and few with type A blood (n = 6), had IRR; compared to type A or AB together, patients with type B (OR 5.00, 95% CI: 1.56-16.06, p = .007) or type O (OR 3.71, 95% CI: 1.44-9.55, p = .007) blood had greater odds of IRR. CONCLUSIONS: This study highlights a possible association of blood type with iv iron IRR; prospective studies with larger patient numbers are warranted to explore this association.

Liver injury in COVID-19 and IL-6 trans-signaling-induced endotheliopathy.

BACKGROUND AND AIMS: COVID-19 is associated with liver injury and elevated interleukin-6 (IL-6). We hypothesized that IL-6 trans-signaling in liver sinusoidal endothelial cells (LSECs) leads to endotheliopathy (a proinflammatory and procoagulant state) and liver injury in COVID-19. METHODS: Coagulopathy, endotheliopathy, and alanine aminotransferase (ALT) were retrospectively analyzed in a subset (n = 68), followed by a larger cohort (n = 3,780) of patients with COVID-19. Liver histology from 43 patients with COVID-19 was analyzed for endotheliopathy and its relationship to liver injury. Primary human LSECs were used to establish the IL-6 trans-signaling mechanism. RESULTS: Factor VIII, fibrinogen, D-dimer, von Willebrand factor (vWF) activity/antigen (biomarkers of coagulopathy/endotheliopathy) were significantly elevated in patients with COVID-19 and liver injury (elevated ALT). IL-6 positively correlated with vWF antigen (p = 0.02), factor VIII activity (p = 0.02), and D-dimer (p <0.0001). On liver histology, patients with COVID-19 and elevated ALT had significantly increased vWF and platelet staining, supporting a link between liver injury, coagulopathy, and endotheliopathy. Intralobular neutrophils positively correlated with platelet (p <0.0001) and vWF (p <0.01) staining, and IL-6 levels positively correlated with vWF staining (p <0.01). IL-6 trans-signaling leads to increased expression of procoagulant (factor VIII, vWF) and proinflammatory factors, increased cell surface vWF (p <0.01), and increased platelet attachment in LSECs. These effects were blocked by soluble glycoprotein 130 (IL-6 trans-signaling inhibitor), the JAK inhibitor ruxolitinib, and STAT1/3 small-interfering RNA knockdown. Hepatocyte fibrinogen expression was increased by the supernatant of LSECs subjected to IL-6 trans-signaling. CONCLUSION: IL-6 trans-signaling drives the coagulopathy and hepatic endotheliopathy associated with COVID-19 and could be a possible mechanism behind liver injury in these patients. LAY SUMMARY: Patients with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection often have liver injury, but why this occurs remains unknown. High levels of interleukin-6 (IL-6) and its circulating receptor, which form a complex to induce inflammatory signals, have been observed in patients with COVID-19. This paper demonstrates that the IL-6 signaling complex causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury.

Low-dose Aspirin prevents hypertension and cardiac fibrosis when thromboxane A2 is unrestrained.

Enhanced platelet activation has been reported in patients with essential hypertension and heart failure. The possible contribution of platelet-derived thromboxane (TX)A2 in their pathophysiology remains unclear. We investigated the systemic TXA2 biosynthesis in vivo and gene expression of its receptor TP in 22 essential hypertension patients and a mouse model of salt-sensitive hypertension. The contribution of platelet TXA2 biosynthesis on enhanced blood pressure (BP) and overload-induced cardiac fibrosis was explored in mice by treating with low-dose Aspirin, resulting in selective inhibition of platelet cyclooxygenase (COX)-1-dependent TXA2 generation. In essential hypertensive patients, systemic biosynthesis of TXA2 [assessed by measuring its urinary metabolites (TXM) reflecting predominant platelet source] was enhanced together with higher gene expression of circulating leukocyte TP and TGF-ß, vs. normotensive controls. Similarly, in hypertensive mice with prostacyclin (PGI2) receptor (IP) deletion (IPKO) fed with a high-salt diet, enhanced urinary TXM, and left ventricular TP overexpression were detected vs. normotensive wildtype (WT) mice. Increased cardiac collagen deposition and profibrotic gene expression (including TGF-ß) was found. Low-dose Aspirin administration caused a selective inhibition of platelet TXA2 biosynthesis and mitigated enhanced blood pressure, cardiac fibrosis, and left ventricular profibrotic gene expression in IPKO but not WT mice. Moreover, the number of myofibroblasts and extravasated platelets in the heart was reduced. In cocultures of human platelets and myofibroblasts, platelet TXA2 induced profibrotic gene expression, including TGF-ß1. In conclusion, our results support tailoring low-dose Aspirin treatment in hypertensive patients with unconstrained TXA2/TP pathway to reduce blood pressure and prevent early cardiac fibrosis.

LMO7 Is a Negative Feedback Regulator of Transforming Growth Factor ß Signaling and Fibrosis.

BACKGROUND: Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor ß (TGF-ß) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-ß1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. METHODS: We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. RESULTS: We demonstrate that LMO7 is induced postinjury and by TGF-ß in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-ß signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-ß treatment. TGF-ß neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-ß signaling by inducing expression of Tgfb1 mRNA, TGF-ß protein, αv and ß3 integrins that promote activation of latent TGF-ß, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1-dependent TGF-ß autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. CONCLUSIONS: LMO7 is induced by TGF-ß and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-ß pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.

Parkin Coordinates Platelet Stress Response in Diabetes Mellitus: A Big Role in a Small Cell.

Increased platelet activation and apoptosis are characteristic of diabetic (DM) platelets, where a Parkin-dependent mitophagy serves a major endogenous protective role. We now demonstrate that Parkin is highly expressed in both healthy platelets and diabetic platelets, compared to other mitochondria-enriched tissues such as the heart, muscle, brain, and liver. Abundance of Parkin in a small, short-lived anucleate cell suggest significance in various key processes. Through proteomics we identified 127 Parkin-interacting proteins in DM platelets and compared them to healthy controls. We assessed the 11 highest covered proteins by individual IPs and confirmed seven proteins that interacted with Parkin; VCP/p97, LAMP1, HADHA, FREMT3, PDIA, ILK, and 14-3-3. Upon further STRING analysis using GO and KEGG, interactions were divided into two broad groups: targeting platelet activation through (1) actions on mitochondria and (2) actions on integrin signaling. Parkin plays an important role in mitochondrial protection through mitophagy (VCP/p97), recruiting phagophores, and targeting lysosomes (with LAMP1). Mitochondrial ß-oxidation may also be regulated by the Parkin/HADHA interaction. Parkin may regulate platelet aggregation and activation through integrin signaling through interactions with proteins like FREMT3, PDIA, ILK, and 14-3-3. Thus, platelet Parkin may regulate the protection (mitophagy) and stress response (platelet activation) in DM platelets. This study identified new potential therapeutic targets for platelet mitochondrial dysfunction and hyperactivation in diabetes mellitus.

Opposing Actions of AKT (Protein Kinase B) Isoforms in Vascular Smooth Muscle Injury and Therapeutic Response.

OBJECTIVE: Drug-eluting stent delivery of mTORC1 (mechanistic target of rapamycin complex 1) inhibitors is highly effective in preventing intimal hyperplasia after coronary revascularization, but adverse effects limit their use for systemic vascular disease. Understanding the mechanism of action may lead to new treatment strategies. We have shown that rapamycin promotes vascular smooth muscle cell differentiation in an AKT2-dependent manner in vitro. Here, we investigate the roles of AKT (protein kinase B) isoforms in intimal hyperplasia. APPROACH AND RESULTS: We found that germ-line-specific or smooth muscle-specific deletion of Akt2 resulted in more severe intimal hyperplasia compared with control mice after arterial denudation injury. Conversely, smooth muscle-specific Akt1 knockout prevented intimal hyperplasia, whereas germ-line Akt1 deletion caused severe thrombosis. Notably, rapamycin prevented intimal hyperplasia in wild-type mice but had no therapeutic benefit in Akt2 knockouts. We identified opposing roles for AKT1 and AKT2 isoforms in smooth muscle cell proliferation, migration, differentiation, and rapamycin response in vitro. Mechanistically, rapamycin induced MYOCD (myocardin) mRNA expression. This was mediated by AKT2 phosphorylation and nuclear exclusion of FOXO4 (forkhead box O4), inhibiting its binding to the MYOCD promoter. CONCLUSIONS: Our data reveal opposing roles for AKT isoforms in smooth muscle cell remodeling. AKT2 is required for rapamycin's therapeutic inhibition of intimal hyperplasia, likely mediated in part through AKT2-specific regulation of MYOCD via FOXO4. Because AKT2 signaling is impaired in diabetes mellitus, this work has important implications for rapamycin therapy, particularly in diabetic patients.

A form of the metabolic syndrome associated with mutations in DYRK1B.

BACKGROUND: Genetic analysis has been successful in identifying causative mutations for individual cardiovascular risk factors. Success has been more limited in mapping susceptibility genes for clusters of cardiovascular risk traits, such as those in the metabolic syndrome. METHODS: We identified three large families with coinheritance of early-onset coronary artery disease, central obesity, hypertension, and diabetes. We used linkage analysis and whole-exome sequencing to identify the disease-causing gene. RESULTS: A founder mutation was identified in DYRK1B, substituting cysteine for arginine at position 102 in the highly conserved kinase-like domain. The mutation precisely cosegregated with the clinical syndrome in all the affected family members and was absent in unaffected family members and unrelated controls. Functional characterization of the disease gene revealed that nonmutant protein encoded by DYRK1B inhibits the SHH (sonic hedgehog) and Wnt signaling pathways and consequently enhances adipogenesis. Furthermore, DYRK1B promoted the expression of the key gluconeogenic enzyme glucose-6-phosphatase. The R102C allele showed gain-of-function activities by potentiating these effects. A second mutation, substituting proline for histidine 90, was found to cosegregate with a similar clinical syndrome in an ethnically distinct family. CONCLUSIONS: These findings indicate a role for DYRK1B in adipogenesis and glucose homeostasis and associate its altered function with an inherited form of the metabolic syndrome. (Funded by the National Institutes of Health.).

Hyperglycemia repression of miR-24 coordinately upregulates endothelial cell expression and secretion of von Willebrand factor.

An elevated level of von Willebrand factor (VWF) in diabetic patients is associated with increased risk of thrombotic cardiovascular events. The underlying mechanism of how VWF expression is upregulated in diabetes mellitus is poorly understood. We now report that hyperglycemia-induced repression of microRNA-24 (miR-24) increases VWF expression and secretion in diabetes mellitus. In diabetic patients and diabetic mouse models (streptozotocin/high-fat diet-induced and db/db mice), miR-24 is reduced in both tissues and plasma. Knockdown of miR-24 in mice leads to increased VWF mRNA and protein levels and enhanced platelet tethering (spontaneous thrombosis). miR-24 tightly controls VWF levels through pleiotropic effects, including direct binding to the 3' untranslated region of VWF and targeting FURIN and the histamine H1 receptor, known regulators of VWF processing and secretion in endothelial cells. We present a novel mechanism for miR-24 downregulation through hyperglycemia-induced activation of aldose reductase, reactive oxygen species, and c-Myc. These findings support a critical role for hyperglycemic repression of miR-24 in VWF-induced pathology. miR-24 represents a novel therapeutic target to prevent adverse thrombotic events in patients with diabetes mellitus.