Fishing for "complements" with vascular organoid models of microvascular disease.

In this issue of Cell Stem Cell, Kawakami et al. develop a SARS-CoV-2 infection-competent, progenitor-derived, human vascular organoid model and uncover a role for complement factor D (CFD) in mediating microvascular immunothrombosis. This model may be applied to conditions where microvascular disease plays a major pathogenic role.

Lipid remodeling in megakaryocyte differentiation and platelet biogenesis.

Lipid remodeling, from fatty acid transport and de novo lipid synthesis, is necessary for megakaryocyte differentiation and platelet production. Dietary saturated fatty acids, impaired fatty acid transport and/or dysfunction in lipid biogenesis can contribute to low platelet counts.

Insights into platelet pharmacology from a cryo-EM structure of the ABCC4 transporter.

Structural determination of the ABCC4 transporter is a major first step in providing crucial molecular insights into the transport of platelet substrates into granules, as well as drug transport from platelets. The findings provide a framework for understanding platelet interactions and potential design of specific platelet antagonists.

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.

Proteomic profiling demonstrates inflammatory and endotheliopathy signatures associated with impaired cardiopulmonary exercise hemodynamic profile in Post Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome.

Approximately 50% of patients who recover from the acute SARS-CoV-2 experience Post Acute Sequelae of SARS-CoV-2 infection (PASC) syndrome. The pathophysiological hallmark of PASC is characterized by impaired system oxygen extraction (EO2) on invasive cardiopulmonary exercise test (iCPET). However, the mechanistic insights into impaired EO2 remain unclear. We studied 21 consecutive iCPET in PASC patients with unexplained exertional intolerance. PASC patients were dichotomized into mildly reduced (EO2peak-mild) and severely reduced (EO2peak-severe) EO2 groups according to the median peak EO2 value. Proteomic profiling was performed on mixed venous blood plasma obtained at peak exercise during iCPET. PASC patients as a group exhibited depressed peak exercise aerobic capacity (peak VO2; 85 ± 18 vs. 131 ± 45% predicted; p = 0.0002) with normal systemic oxygen delivery, DO2 (37 ± 9 vs. 42 ± 15 mL/kg/min; p = 0.43) and reduced EO2 (0.4 ± 0.1 vs. 0.8 ± 0.1; p < 0.0001). PASC patients with EO2peak-mild exhibited greater DO2 compared to those with EO2peak-severe [42.9 (34.2-41.2) vs. 32.1 (26.8-38.0) mL/kg/min; p = 0.01]. The proteins with increased expression in the EO2peak-severe group were involved in inflammatory and fibrotic processes. In the EO2peak-mild group, proteins associated with oxidative phosphorylation and glycogen metabolism were elevated. In PASC patients with impaired EO2, there exist a spectrum of PASC phenotype related to differential aberrant protein expression and cardio-pulmonary physiologic response. PASC patients with EO2peak-severe exhibit a maladaptive physiologic and proteomic signature consistent with persistent inflammatory state and endothelial dysfunction, while in the EO2peak-mild group, there is enhanced expression of proteins involved in oxidative phosphorylation-mediated ATP synthesis along with an enhanced cardiopulmonary physiological response.

The age of bone marrow dictates the clonality of smooth muscle-derived cells in atherosclerotic plaques.

Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to ~70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin ß3 resulting in increased tumor necrosis factor [TNF]-α signaling. TNFα signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin ß3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

Platelet-derived TLT-1 promotes tumor progression by suppressing CD8+ T cells.

Current understanding of tumor immunosuppressive mechanisms forms the basis for modern day immunotherapies. Immunoregulatory role of platelets in cancer remains largely elusive. Platelets from non-small cell lung cancer (NSCLC) patients revealed a distinct activation phenotype. TREM-like transcript 1 (TLT-1), a platelet protein, was increased along with enhanced extracellular release from NSCLC platelets. The increased platelet TLT-1 was also evident in humanized mice with patient-derived tumors. In immunocompetent mice with syngeneic tumors, TLT-1 binding to T cells, in vivo, led to suppression of CD8 T cells, promoting tumor growth. We identified direct interaction between TLT-1 and CD3ε on T cells, implicating the NF-κB pathway in CD8 T cell suppression. Anti-TLT-1 antibody rescued patients' T cells from platelet-induced suppression ex vivo and reduced tumors in mice in vivo. Clinically, higher TLT-1 correlated with reduced survival of NSCLC patients. Our findings thus identify TLT-1 as a platelet-derived immunosuppressor that suppresses CD8 T cells and demonstrate its therapeutic and prognostic significance in cancer.

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.

Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis.

Platelets have emerged as key inflammatory cells implicated in the pathology of sepsis, but their contributions to rapid clinical deterioration and dysregulated inflammation have not been defined. Here, we show that the incidence of thrombocytopathy and inflammatory cytokine release was significantly increased in patients with severe sepsis. Platelet proteomic analysis revealed significant upregulation of gasdermin D (GSDMD). Using platelet-specific Gsdmd-deficient mice, we demonstrated a requirement for GSDMD in triggering platelet pyroptosis in cecal ligation and puncture (CLP)-induced sepsis. GSDMD-dependent platelet pyroptosis was induced by high levels of S100A8/A9 targeting toll-like receptor 4 (TLR4). Pyroptotic platelet-derived oxidized mitochondrial DNA (ox-mtDNA) potentially promoted neutrophil extracellular trap (NET) formation, which contributed to platelet pyroptosis by releasing S100A8/A9, forming a positive feedback loop that led to the excessive release of inflammatory cytokines. Both pharmacological inhibition using Paquinimod and genetic ablation of the S100A8/A9-TLR4 signaling axis improved survival in mice with CLP-induced sepsis by suppressing platelet pyroptosis.

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.

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.

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.

A guide to molecular and functional investigations of platelets to bridge basic and clinical sciences.

Platelets have been shown to be associated with pathophysiological process beyond thrombosis, demonstrating critical additional roles in homeostatic processes, such as immune regulation, and vascular remodeling. Platelets themselves can have multiple functional states and can communicate and regulate other cells including immune cells and vascular smooth muscle cells, to serve such diverse functions. Although traditional platelet functional assays are informative and reliable, they are limited in their ability to unravel platelet phenotypic heterogeneity and interactions. Developments in methods such as electron microscopy, flow cytometry, mass spectrometry, and 'omics' studies, have led to new insights. In this Review, we focus on advances in platelet biology and function, with an emphasis on current and promising methodologies. We also discuss technical and biological challenges in platelet investigations. Using coronavirus disease 2019 (COVID-19) as an example, we further describe the translational relevance of these approaches and the possible 'bench-to-bedside' utility in patient diagnosis and care.

Gasdermin D inhibition confers antineutrophil-mediated cardioprotection in acute myocardial infarction.

Acute myocardial infarction (AMI) induces blood leukocytosis, which correlates inversely with patient survival. The molecular mechanisms leading to leukocytosis in the infarcted heart remain poorly understood. Using an AMI mouse model, we identified gasdermin D (GSDMD) in activated leukocytes early in AMI. We demonstrated that GSDMD is required for enhanced early mobilization of neutrophils to the infarcted heart. Loss of GSDMD resulted in attenuated IL-1ß release from neutrophils and subsequent decreased neutrophils and monocytes in the infarcted heart. Knockout of GSDMD in mice significantly reduced infarct size, improved cardiac function, and increased post-AMI survival. Through a series of bone marrow transplantation studies and leukocyte depletion experiments, we further clarified that excessive bone marrow-derived and GSDMD-dependent early neutrophil production and mobilization (24 hours after AMI) contributed to the detrimental immunopathology after AMI. Pharmacological inhibition of GSDMD also conferred cardioprotection after AMI through a reduction in scar size and enhancement of heart function. Our study provides mechanistic insights into molecular regulation of neutrophil generation and mobilization after AMI, and supports GSDMD as a new target for improved ventricular remodeling and reduced heart failure after AMI.

Cardioprotective mechanism of SGLT2 inhibitor against myocardial infarction is through reduction of autosis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with diabetes mellitus but the protective mechanism remains elusive. Here we demonstrated that the SGLT2 inhibitor, Empagliflozin (EMPA), suppresses cardiomyocytes autosis (autophagic cell death) to confer cardioprotective effects. Using myocardial infarction (MI) mouse models with and without diabetes mellitus, EMPA treatment significantly reduced infarct size, and myocardial fibrosis, thereby leading to improved cardiac function and survival. In the context of ischemia and nutritional glucose deprivation where autosis is already highly stimulated, EMPA directly inhibits the activity of the Na+/H+ exchanger 1 (NHE1) in the cardiomyocytes to regulate excessive autophagy. Knockdown of NHE1 significantly rescued glucose deprivation-induced autosis. In contrast, overexpression of NHE1 aggravated the cardiomyocytes death in response to starvation, which was effectively rescued by EMPA treatment. Furthermore, in vitro and in vivo analysis of NHE1 and Beclin 1 knockout mice validated that EMPA's cardioprotective effects are at least in part through downregulation of autophagic flux. These findings provide new insights for drug development, specifically targeting NHE1 and autosis for ventricular remodeling and heart failure after MI in both diabetic and non-diabetic patients.