Event Title
Role of CRK and CRKL Adaptor Proteins in Vascular Endothelial Cell Biology
Location
Philadelphia, PA
Start Date
11-5-2022 1:00 PM
End Date
11-5-2022 4:00 PM
Description
Introduction: Proper formation of the coronary vessels is crucial during cardiovascular development. The coronary vessels are important for delivering oxygen and nutrients to the underlying myocardium. However, the molecular and cellular mechanisms that contribute to coronary vessel formation are poorly understood. Our lab identified Reelin, an extracellular matrix glycoprotein encoded by the RELN gene, as a potential contributor to vasculogenic and angiogenic processes during mammalian heart development. The Reelin functions through a signaling cascade that leads to activation of downstream effectors. Two of these effectors are the adaptor proteins Chicken tumor virus no. 10 Regulator of Kinase (CRK) and its paralog CRK like (CRKL). We observed Reelin to be localized to the vascular endothelial cells lining nascent and mature blood vessels in the embryonic mouse heart. We demonstrated that gene silencing of RELN in primary cultures of human dermal microvascular endothelial cells (HDMECs) via small interfering RNAs (siRNA) led to changes in cell biology. These changes included a reduction in vascular endothelial cell migration, a reduction in cell membrane permeability and an increase in capillary-like tube formation. At the molecular level, silencing of RELN altered expression of the angiogenic transcripts Angiopoietin-2 (ANGPT2) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 1 (ADAMTS1). Together these findings led us to speculate how Reelin modulates vascular endothelial cells.
Objectives: Based on our understanding of the Reelin signaling pathway, we established an objective to examine the role of the CRK and CRKL downstream effectors in mediating these biological functions of Reeln in vascular endothelial cells. We hypothesized that CRK and CRKL may induce changes at the molecular level to angiogenic transcripts to mediate the biological functions of Reelin in vascular endothelial cells.
Methods: We conducted experiments on HDMECs because they serve as an in vitro model to examine the contribution of CRK and CRKL to expression of angiogenic markers and cell morphology in vascular endothelial cells. Gene-specific siRNAs were used to target endogenous expression of CRK and CRKL in HDMECs. We analyzed ANGPT2 and ADAMTS1 mRNA and protein expression in control and CRK/CRKL knockdown (KD) cells by Taqman qPCR and western blot analyses, respectively. Also, we analyzed the morphological characteristics of these cells.
Results: siRNA-mediated knockdown of CRK/CRKL KD in HDMECs yielded almost a 90% and a 75% reduction of CRK and CRKL mRNA expression relative to negative control cells. The CRK/CRKL KD HDMECs took on an irregularly elongated morphology with reduced density in comparison to the negative control cells. ANGPT2 mRNA expression was significantly downregulated by nearly 70%, and ADAMTS1 protein expression appeared to be upregulated in CRK/CRKL KD cells versus control cells. In addition, RELN mRNA expression was unaffected by silencing of CRK and CRKL in these cells.
Conclusions: We concluded that CRK and CRKL influence vascular endothelial cell morphology and the expression of the angiogenic transcripts, ANGPT2 and ADAMTS1. Knockdown of CRK/CRKL in HDMECs produced irregularly elongated cells, decreased expression of ANGPT2 mRNA and increased ADAMTS1 protein expression. We observed similar molecular findings with our previous analysis of RELN KD in HDMECs. Altogether these findings provide evidence to support a role for CRK and CRKL as angiogenic molecular mediators of Reelin in vascular endothelial cells.
Embargo Period
5-24-2022
Role of CRK and CRKL Adaptor Proteins in Vascular Endothelial Cell Biology
Philadelphia, PA
Introduction: Proper formation of the coronary vessels is crucial during cardiovascular development. The coronary vessels are important for delivering oxygen and nutrients to the underlying myocardium. However, the molecular and cellular mechanisms that contribute to coronary vessel formation are poorly understood. Our lab identified Reelin, an extracellular matrix glycoprotein encoded by the RELN gene, as a potential contributor to vasculogenic and angiogenic processes during mammalian heart development. The Reelin functions through a signaling cascade that leads to activation of downstream effectors. Two of these effectors are the adaptor proteins Chicken tumor virus no. 10 Regulator of Kinase (CRK) and its paralog CRK like (CRKL). We observed Reelin to be localized to the vascular endothelial cells lining nascent and mature blood vessels in the embryonic mouse heart. We demonstrated that gene silencing of RELN in primary cultures of human dermal microvascular endothelial cells (HDMECs) via small interfering RNAs (siRNA) led to changes in cell biology. These changes included a reduction in vascular endothelial cell migration, a reduction in cell membrane permeability and an increase in capillary-like tube formation. At the molecular level, silencing of RELN altered expression of the angiogenic transcripts Angiopoietin-2 (ANGPT2) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 1 (ADAMTS1). Together these findings led us to speculate how Reelin modulates vascular endothelial cells.
Objectives: Based on our understanding of the Reelin signaling pathway, we established an objective to examine the role of the CRK and CRKL downstream effectors in mediating these biological functions of Reeln in vascular endothelial cells. We hypothesized that CRK and CRKL may induce changes at the molecular level to angiogenic transcripts to mediate the biological functions of Reelin in vascular endothelial cells.
Methods: We conducted experiments on HDMECs because they serve as an in vitro model to examine the contribution of CRK and CRKL to expression of angiogenic markers and cell morphology in vascular endothelial cells. Gene-specific siRNAs were used to target endogenous expression of CRK and CRKL in HDMECs. We analyzed ANGPT2 and ADAMTS1 mRNA and protein expression in control and CRK/CRKL knockdown (KD) cells by Taqman qPCR and western blot analyses, respectively. Also, we analyzed the morphological characteristics of these cells.
Results: siRNA-mediated knockdown of CRK/CRKL KD in HDMECs yielded almost a 90% and a 75% reduction of CRK and CRKL mRNA expression relative to negative control cells. The CRK/CRKL KD HDMECs took on an irregularly elongated morphology with reduced density in comparison to the negative control cells. ANGPT2 mRNA expression was significantly downregulated by nearly 70%, and ADAMTS1 protein expression appeared to be upregulated in CRK/CRKL KD cells versus control cells. In addition, RELN mRNA expression was unaffected by silencing of CRK and CRKL in these cells.
Conclusions: We concluded that CRK and CRKL influence vascular endothelial cell morphology and the expression of the angiogenic transcripts, ANGPT2 and ADAMTS1. Knockdown of CRK/CRKL in HDMECs produced irregularly elongated cells, decreased expression of ANGPT2 mRNA and increased ADAMTS1 protein expression. We observed similar molecular findings with our previous analysis of RELN KD in HDMECs. Altogether these findings provide evidence to support a role for CRK and CRKL as angiogenic molecular mediators of Reelin in vascular endothelial cells.