Shirin Bhaloo - Academia.edu (original) (raw)

Papers by Shirin Bhaloo

Cellular and Molecular Life Sciences, 2020

Stem/progenitor cells (SPCs) have been implicated to participate in vascular repair. However, the... more Stem/progenitor cells (SPCs) have been implicated to participate in vascular repair. However, the exact role of SPCs in endothelial repair of large vessels still remains controversial. This study aimed to delineate the cellular heterogeneity and possible functional role of endogenous vascular SPCs in large vessels. Using single-cell RNA-sequencing (scRNA-seq) and genetic lineage tracing mouse models, we uncovered the cellular heterogeneity of SPCs, i.e., c-Kit + cells in the mouse aorta, and found that endogenous c-Kit + cells acquire endothelial cell fate in the aorta under both physiological and pathological conditions. While c-Kit + cells contribute to aortic endothelial turnover in the atheroprone regions during homeostasis, recipient c-Kit + cells of nonbone marrow source replace both luminal and microvessel endothelial cells in transplant arteriosclerosis. Single-cell pseudotime analysis of scRNA-seq data and in vitro cell experiments suggest that vascular SPCs display endothelial differentiation potential and undergo metabolic reprogramming during cell differentiation, in which AKT/mTOR-dependent glycolysis is critical for endothelial gene expression. These findings demonstrate a critical role for c-Kit lineage cells in aortic endothelial turnover and replacement, and may provide insights into therapeutic strategies for vascular diseases.

Arteriosclerosis, Thrombosis, and Vascular Biology, 2019

Objective: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeosta... more Objective: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-β1 (transforming growth factor β1) and transfection of microRNA (miR)-...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2018

Background: Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by ... more Background: Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by promoting smooth muscle cell (SMC) differentiation and endothelial repair, two processes in which vascular stem/progenitor cells could be involved. However, the effect of Dkk3 on stem/progenitor cell migration and its specific receptor on these cells remain unknown. Methods: Vascular stem/progenitor cells (VPCs) were isolated from murine aortic adventitia and selected for the Sca-1 marker. The chemotactic ability of Dkk3 for Sca-1+ cells was tested in vitro using transwell and wound healing assays and ex vivo by the aortic ring assay. Chemokine receptors identification was carried out using Western blot (WB), siRNA-mediated knockdown, co-Immunoprecipitation (Co-IP) and saturation binding assays. To assess in vivo the role of Dkk3 in recruiting Sca-1+ cells, tissue-engineered vessel grafts, with or without Dkk3, were fabricated and implanted to replace the rat abdominal aorta. Results: We...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2018

Objective— Vascular adventitial Sca1 + (stem cell antigen-1) progenitor cells preferentially diff... more Objective— Vascular adventitial Sca1 + (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1 + cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease. Approach and Results— We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1 + cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1 + adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin , Flk-1 , and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regula...

Journal of Biological Chemistry, 2018

Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical setti... more Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-β1 (TGFβ1), human umbilical cord–derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo. More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indic...

Circulation research, Jan 6, 2018

Vascular progenitor cells (VPCs) play key roles in physiological and pathological vascular remode... more Vascular progenitor cells (VPCs) play key roles in physiological and pathological vascular remodelling, a process that is crucial for the regeneration of acellular biodegradable scaffolds engineered as vital strategies against the limited availability of healthy autologous vessels for bypass grafting. Therefore, understanding the mechanisms driving VPCs recruitment and differentiation could help the development of new strategies to improve Tissue-Engineered Vessel Grafts (TEVGs) and design drug-targeted therapy for vessel regeneration. In this study we sought to investigate the role of Dickkopf-3 (Dkk3), recently identified as a cytokine promotor of endothelial repair and smooth muscle cell differentiation, on VPC cell migration and vascular regeneration and to identify its functional receptor that remains unknown. Vascular stem/progenitor cells were isolated from murine aortic adventitia and selected for the Stem cell antigen-1 (Sca-1) marker. Dkk3 induced the chemotaxis of Sca-1+ ...

Circulation research, Jan 25, 2018

Vascular, resident stem cells are present in all 3 layers of the vessel wall; they play a role in... more Vascular, resident stem cells are present in all 3 layers of the vessel wall; they play a role in vascular formation under physiological conditions and in remodeling in pathological situations. Throughout development and adult early life, resident stem cells participate in vessel formation through vasculogenesis and angiogenesis. In adults, the vascular stem cells are mostly quiescent in their niches but can be activated in response to injury and participate in endothelial repair and smooth muscle cell accumulation to form neointima. However, delineation of the characteristics and of the migration and differentiation behaviors of these stem cells is an area of ongoing investigation. A set of genetic mouse models for cell lineage tracing has been developed to specifically address the nature of these cells and both migration and differentiation processes during physiological angiogenesis and in vascular diseases. This review summarizes the current knowledge on resident stem cells, whi...

Circulation, Jan 26, 2018

BACKGROUND: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of card... more BACKGROUND: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers, such as the cardiosphere-derived cells, or with markers such as Sca1 + , Bmi1 + , Isl1 + , or Abcg2 + cardiac stem cells have been reported. Moreover, it remains unclear whether putative cardiac stem cells with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. METHODS: To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all nonmyocyte populations that contain putative cardiac stem cells. Using dual lineage tracing system, we assessed whether nonmyocytes generated any new myocytes during embryonic development, during adult homeostasis, and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocyte generation from nonmyocytes. RESULTS: By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from nonmyocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from nonmyocytes in the skeletal muscle after injury. CONCLUSIONS: This study provides in vivo genetic evidence for nonmyocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

Arteriosclerosis, Thrombosis, and Vascular Biology, 2017

Objective— Leptin is an adipokine initially thought to be a metabolic factor. Recent publications... more Objective— Leptin is an adipokine initially thought to be a metabolic factor. Recent publications have shown its roles in inflammation and vascular disease, to which Sca-1 + vascular progenitor cells within the vessel wall may contribute. We sought to elucidate the effects of leptin on Sca-1 + progenitor cells migration and neointimal formation and to understand the underlying mechanisms. Approach and Results— Sca-1 + progenitor cells from the vessel wall of Lepr +/+ and Lepr −/− mice were cultured and purified. The migration of Lepr +/+ Sca-1 + progenitor cells in vitro was markedly induced by leptin. Western blotting and kinase assays revealed that leptin induced the activation of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal–regulated kinases 1/2, pFAK (phosphorylated focal adhesion kinase), and Rac1 (ras-related C3 botulinum toxin substrate 1)/Cdc42 (cell division control protein 42 homolog). In a mouse femoral artery guid...

Stem cells (Dayton, Ohio), Jan 11, 2016

Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital ... more Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell-based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation towards a SMC lineage. ESCs were seeded on collagen-IV in differentiation medium to generate ESC-derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4-methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathw...

Cellular and Molecular Life Sciences, 2020

Stem/progenitor cells (SPCs) have been implicated to participate in vascular repair. However, the... more Stem/progenitor cells (SPCs) have been implicated to participate in vascular repair. However, the exact role of SPCs in endothelial repair of large vessels still remains controversial. This study aimed to delineate the cellular heterogeneity and possible functional role of endogenous vascular SPCs in large vessels. Using single-cell RNA-sequencing (scRNA-seq) and genetic lineage tracing mouse models, we uncovered the cellular heterogeneity of SPCs, i.e., c-Kit + cells in the mouse aorta, and found that endogenous c-Kit + cells acquire endothelial cell fate in the aorta under both physiological and pathological conditions. While c-Kit + cells contribute to aortic endothelial turnover in the atheroprone regions during homeostasis, recipient c-Kit + cells of nonbone marrow source replace both luminal and microvessel endothelial cells in transplant arteriosclerosis. Single-cell pseudotime analysis of scRNA-seq data and in vitro cell experiments suggest that vascular SPCs display endothelial differentiation potential and undergo metabolic reprogramming during cell differentiation, in which AKT/mTOR-dependent glycolysis is critical for endothelial gene expression. These findings demonstrate a critical role for c-Kit lineage cells in aortic endothelial turnover and replacement, and may provide insights into therapeutic strategies for vascular diseases.

Arteriosclerosis, Thrombosis, and Vascular Biology, 2019

Objective: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeosta... more Objective: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-β1 (transforming growth factor β1) and transfection of microRNA (miR)-...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2018

Background: Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by ... more Background: Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by promoting smooth muscle cell (SMC) differentiation and endothelial repair, two processes in which vascular stem/progenitor cells could be involved. However, the effect of Dkk3 on stem/progenitor cell migration and its specific receptor on these cells remain unknown. Methods: Vascular stem/progenitor cells (VPCs) were isolated from murine aortic adventitia and selected for the Sca-1 marker. The chemotactic ability of Dkk3 for Sca-1+ cells was tested in vitro using transwell and wound healing assays and ex vivo by the aortic ring assay. Chemokine receptors identification was carried out using Western blot (WB), siRNA-mediated knockdown, co-Immunoprecipitation (Co-IP) and saturation binding assays. To assess in vivo the role of Dkk3 in recruiting Sca-1+ cells, tissue-engineered vessel grafts, with or without Dkk3, were fabricated and implanted to replace the rat abdominal aorta. Results: We...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2018

Objective— Vascular adventitial Sca1 + (stem cell antigen-1) progenitor cells preferentially diff... more Objective— Vascular adventitial Sca1 + (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1 + cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease. Approach and Results— We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1 + cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1 + adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin , Flk-1 , and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regula...

Journal of Biological Chemistry, 2018

Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical setti... more Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-β1 (TGFβ1), human umbilical cord–derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo. More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indic...

Circulation research, Jan 6, 2018

Vascular progenitor cells (VPCs) play key roles in physiological and pathological vascular remode... more Vascular progenitor cells (VPCs) play key roles in physiological and pathological vascular remodelling, a process that is crucial for the regeneration of acellular biodegradable scaffolds engineered as vital strategies against the limited availability of healthy autologous vessels for bypass grafting. Therefore, understanding the mechanisms driving VPCs recruitment and differentiation could help the development of new strategies to improve Tissue-Engineered Vessel Grafts (TEVGs) and design drug-targeted therapy for vessel regeneration. In this study we sought to investigate the role of Dickkopf-3 (Dkk3), recently identified as a cytokine promotor of endothelial repair and smooth muscle cell differentiation, on VPC cell migration and vascular regeneration and to identify its functional receptor that remains unknown. Vascular stem/progenitor cells were isolated from murine aortic adventitia and selected for the Stem cell antigen-1 (Sca-1) marker. Dkk3 induced the chemotaxis of Sca-1+ ...

Circulation research, Jan 25, 2018

Vascular, resident stem cells are present in all 3 layers of the vessel wall; they play a role in... more Vascular, resident stem cells are present in all 3 layers of the vessel wall; they play a role in vascular formation under physiological conditions and in remodeling in pathological situations. Throughout development and adult early life, resident stem cells participate in vessel formation through vasculogenesis and angiogenesis. In adults, the vascular stem cells are mostly quiescent in their niches but can be activated in response to injury and participate in endothelial repair and smooth muscle cell accumulation to form neointima. However, delineation of the characteristics and of the migration and differentiation behaviors of these stem cells is an area of ongoing investigation. A set of genetic mouse models for cell lineage tracing has been developed to specifically address the nature of these cells and both migration and differentiation processes during physiological angiogenesis and in vascular diseases. This review summarizes the current knowledge on resident stem cells, whi...

Circulation, Jan 26, 2018

BACKGROUND: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of card... more BACKGROUND: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers, such as the cardiosphere-derived cells, or with markers such as Sca1 + , Bmi1 + , Isl1 + , or Abcg2 + cardiac stem cells have been reported. Moreover, it remains unclear whether putative cardiac stem cells with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. METHODS: To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all nonmyocyte populations that contain putative cardiac stem cells. Using dual lineage tracing system, we assessed whether nonmyocytes generated any new myocytes during embryonic development, during adult homeostasis, and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocyte generation from nonmyocytes. RESULTS: By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from nonmyocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from nonmyocytes in the skeletal muscle after injury. CONCLUSIONS: This study provides in vivo genetic evidence for nonmyocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

Arteriosclerosis, Thrombosis, and Vascular Biology, 2017

Objective— Leptin is an adipokine initially thought to be a metabolic factor. Recent publications... more Objective— Leptin is an adipokine initially thought to be a metabolic factor. Recent publications have shown its roles in inflammation and vascular disease, to which Sca-1 + vascular progenitor cells within the vessel wall may contribute. We sought to elucidate the effects of leptin on Sca-1 + progenitor cells migration and neointimal formation and to understand the underlying mechanisms. Approach and Results— Sca-1 + progenitor cells from the vessel wall of Lepr +/+ and Lepr −/− mice were cultured and purified. The migration of Lepr +/+ Sca-1 + progenitor cells in vitro was markedly induced by leptin. Western blotting and kinase assays revealed that leptin induced the activation of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal–regulated kinases 1/2, pFAK (phosphorylated focal adhesion kinase), and Rac1 (ras-related C3 botulinum toxin substrate 1)/Cdc42 (cell division control protein 42 homolog). In a mouse femoral artery guid...

Stem cells (Dayton, Ohio), Jan 11, 2016

Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital ... more Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell-based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation towards a SMC lineage. ESCs were seeded on collagen-IV in differentiation medium to generate ESC-derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4-methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathw...