Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

BACKGROUND: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. METHODS: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. RESULTS: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. CONCLUSIONS: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Abcg2-expressing side population cells contribute to cardiomyocyte renewal through fusion.

The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

BACKGROUND: Although cardiac c-kit+ cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit+ cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit+ cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit+ cells. METHODS: We used single-cell sequencing and genetic lineage tracing of c-kit+ cells to determine whether various pathological stimuli would result in different fates of c-kit+ cells. RESULTS: Single-cell sequencing of cardiac CD45-c-kit+ cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit+ cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit+ cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. CONCLUSIONS: These results demonstrate that different pathological stimuli induce different cell fates of c-kit+ cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit+ cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit+ cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit+ cells.

Hypoxia-induced cardiac injury in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a disease of progressive destruction of striated muscle, resulting in muscle weakness with progressive respiratory and cardiac failure. Respiratory and cardiac disease are the leading causes of death in DMD patients. Previous studies have suggested an important link between cardiac dysfunction and hypoxia in the dystrophic heart; these studies aim to understand the mechanism underlying this connection. Here we demonstrate that anesthetized dystrophic mice display significant mortality following acute exposure to hypoxia. This increased mortality is associated with a significant metabolic acidosis, despite having significantly higher levels of arterial Po2 Chronic hypoxia does not result in mortality, but rather is characterized by marked cardiac fibrosis. Studies in isolated hearts reveal that the contractile function of dystrophic hearts is highly susceptible to short bouts of ischemia, but these hearts tolerate prolonged acidosis better than wild-type hearts, indicating an increased sensitivity of the dystrophic heart to hypoxia. Dystrophic hearts display decreased cardiac efficiency and oxygen extraction. Isolated dystrophic cardiomyocytes and hearts have normal levels of FCCP-induced oxygen consumption, and mitochondrial morphology and content are normal in the dystrophic heart. These studies demonstrate reductions in cardiac efficiency and oxygen extraction of the dystrophic heart. The underlying cause of this reduced oxygen extraction is not clear; however, the current studies suggest that large disruptions of mitochondrial respiratory function or coronary flow regulation are not responsible. This finding is significant, as hypoxia is a common and largely preventable component of DMD that may contribute to the progression of the cardiac disease in DMD patients.

The Role of Cardiac Side Population Cells in Cardiac Regeneration.

The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies.

Secreted osteopontin is highly polymerized in human airways and fragmented in asthmatic airway secretions.

BACKGROUND: Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family and a cytokine with diverse biologic roles. OPN undergoes extensive post-translational modifications, including polymerization and proteolytic fragmentation, which alters its biologic activity. Recent studies suggest that OPN may contribute to the pathogenesis of asthma. METHODOLOGY: To determine whether secreted OPN (sOPN) is polymerized in human airways and whether it is qualitatively different in asthma, we used immunoblotting to examine sOPN in bronchoalveolar lavage (BAL) fluid samples from 12 healthy and 21 asthmatic subjects (and in sputum samples from 27 healthy and 21 asthmatic subjects). All asthmatic subjects had mild to moderate asthma and abstained from corticosteroids during the study. Furthermore, we examined the relationship between airway sOPN and cellular inflammation. PRINCIPAL FINDINGS: We found that sOPN in BAL fluid and sputum exists in polymeric, monomeric, and cleaved forms, with most of it in polymeric form. Compared to healthy subjects, asthmatic subjects had proportionately less polymeric sOPN and more monomeric and cleaved sOPN. Polymeric sOPN in BAL fluid was associated with increased alveolar macrophage counts in airways in all subjects. CONCLUSIONS: These results suggest that sOPN in human airways (1) undergoes extensive post-translational modification by polymerization and proteolytic fragmentation, (2) is more fragmented and less polymerized in subjects with mild to moderate asthma, and (3) may contribute to recruitment or survival of alveolar macrophages.