ALK1 Signaling in Human Cardiac Progenitor Cells Promotes a Pro-angiogenic Secretome.

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Activin A receptor-like type 1 (ACVRL1, ALK1) signaling plays a pivotal role in cardiovascular development and maintenance, but its importance in human-derived therapeutic cardiac cells is not well understood. Here, we isolated a subpopulation of human highly proliferative cells (hHiPCs) from adult epicardial tissue and found that they express ALK1, a high affinity receptor for bone morphogenetic protein-9 (BMP9), which signals via SMAD1/5 to regulate paracrine/autocrine signaling and angiogenesis. We show that in humans, circulating BMP9 level is negatively associated with the number of epicardial hHiPC and positively associated with endothelial cell (EC) number in the adult heart, implicating the potential importance of this signaling pathway in cardiac cell fate and vascular maintenance. To investigate BMP9/ALK1 signaling in hHiPCs, we selected a primary cell population of hHiPC from each of 3 individuals and studied their responses to BMP9 and BMP10 treatment in vitro. Proteins were collected in conditioned media (CM) for mass spectrometry and cell-based assays on human ECs and hHiPCs. Proteomic analysis of the hHiPC secretome following BMP9 or BMP10 treatment demonstrates that the secreted proteins, sclerostin (SOST), meflin/immunoglobulin superfamily containing leucine rich repeat (ISLR), and insulin-like growth factor binding protein-3 (IGFBP3), are novel regulated targets of BMP9/ALK1 signaling. Lentiviral shRNA and pharmacological inhibition of ALK1 in hHiPCs suppressed transcription and secretion of SOST, ISLR, and IGFBP3 following BMP9 treatment. Moreover, the BMP9-treated secretome of hHiPC increased capillary-like tube formation of ECs and hHiPCs. Treatment of hHiPCs with recombinant SOST increased VEGF-a expression, increased tube formation and enhanced expression of EC receptor marker annexin A2 (ANXA2). These data provide the first proteomic characterization of hHiPC, identifying BMP9/ALK1-mediated target protein secretion in hHiPCs, and underscore the complex role of BMP9/ALK1 signaling in paracrine/autocrine mediated angiogenesis. 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Proteomic analysis of the hHiPC secretome following BM", "xin A2 (ANXA2). These data provide the first proteomic characterization of hHiPC, identifying BMP9/"], "order_basis": "paper_text_order:1179", "id": "assay:1"}, {"role": "data_generation", "definition_id": "transfection", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["d targets of BMP9/ALK1 signaling. Lentiviral shRNA and pharmacological inhibition of ALK1 in hH", "Materials and Methods Cell culture, shRNA transfection, and in vitro stimulation hHiPCs were isolat", "Materials and Methods Cell culture, shRNA transfection, and in vitro stimulation hHiPC"], "order_basis": "paper_text_order:1575", "id": "assay:2"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.77, "evidence": ["lial cells (HMEC-1, ATCC, Manassas, VA) were cultured in EGM2 media at 37°C in 5% CO2 and used under"], "order_basis": "paper_text_order:3385", "id": "assay:3"}, {"role": "data_generation", "definition_id": "qpcr", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["sis of BMP9 target secreted proteins we used RT-qPCR analysis to determine transcriptional regula", "n BMP9-target CD105 (positive control) [55]. RT-qPCR measurements also confirmed upregulation of", "regulation of secretome proteins by BMP9.A) RT-qPCR of hHiPC (n=5 independent patient-derived cl", "xpression for SOST, IGFBP3, ISLR, and CXCL5. RT-qPCR responses were normalized to 18S. 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