Novel Mediators of Reelin Signaling in Human Vascular Endothelial Cells
Location
Philadelphia, PA
Start Date
11-5-2022 1:00 PM
End Date
11-5-2022 4:00 PM
Description
Introduction: The formation of coronary vessels is fundamental in heart development. Our lab has demonstrated that Reelin, an extracellular matrix glycoprotein, is involved in the formation of coronary vessels. Reelin expression is localized to the vascular endothelial cells of coronary vessels in embryonic mice. Human dermal microvascular endothelial cells (HDMECs) were used to examine the potential role of Reelin in vascular development as they appropriately model events involved in vessel formation and endogenously express RELN mRNA. Previous studies from our lab indicated that small interfering RNA (siRNA)-mediated gene silencing of RELN in HDMECs reduced cell migration and basal cell membrane permeability, and increased capillary-like tube formation. Based on these results, we believe Reelin is involved in vasculogenesis and angiogenesis. This prompted us to analyze expression of angiogenic transcripts by qPCR array in negative control and RELN knockdown (KD) HDMECs. Our results indicated significant upregulation of ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs), and significant changes in expression of other angiogenesis-related transcripts.
Study Objective: This study aimed to validate the angiogenesis array data by individual TaqMan qPCR analysis of mRNA expression of the significantly altered transcripts. Also, we aimed to analyze the encoded protein expression of these transcripts in negative control and RELN KD cells. We hypothesized that significant alterations in expression of ADAMTS1 and other angiogenic transcripts leads to changes in their protein expression in our RELN KD cells and mediates Reelin signaling in human vascular endothelial cells.
Methods: To validate our angiogenesis qPCR array data, we utilized HDMECs as an in vitro system to examine mRNA and protein expression. Gene-specific siRNAs were used to target and knockdown endogenous expression of RELN in HDMECs. Total RNA was isolated from negative control and RELN KD HDMECs and it was analyzed by qPCR with TaqMan probes for RELN, ADAMTS1, GAPDH, 18SRNA and other angiogenic transcripts. Also, protein expression of the encoded transcripts was analyzed by immunofluorescence and western blot in negative control and RELN KD HDMECs.
Results: RELN KD HDMECs exhibited a >90% reduction in Reelin protein expression compared to negative control HDMECs. ADAMTS1 mRNA was significantly upregulated in RELN KD cells while expression of other angiogenic transcripts were also altered in these cells. We demonstrated by immunofluorescence that ADAMTS1 is localized to the nucleus, and it is detected in HDMEC lysates by western blot analysis. In addition, ADAMTS1 protein expression differed between negative control and RELN KD HDMECs.
Conclusions: We conclude that ADAMTS1 mRNA is upregulated in RELN KD HDMECs versus negative control cells. Knockdown of RELN in HDMECS altered cell migration, membrane permeability, capillary-like tube formation and expression of angiogenic transcripts. These findings provide key evidence that ADAMTS1 and other angiogenic transcripts may be novel mediators of Reelin signaling in human vascular endothelial cells.
Embargo Period
5-24-2022
Novel Mediators of Reelin Signaling in Human Vascular Endothelial Cells
Philadelphia, PA
Introduction: The formation of coronary vessels is fundamental in heart development. Our lab has demonstrated that Reelin, an extracellular matrix glycoprotein, is involved in the formation of coronary vessels. Reelin expression is localized to the vascular endothelial cells of coronary vessels in embryonic mice. Human dermal microvascular endothelial cells (HDMECs) were used to examine the potential role of Reelin in vascular development as they appropriately model events involved in vessel formation and endogenously express RELN mRNA. Previous studies from our lab indicated that small interfering RNA (siRNA)-mediated gene silencing of RELN in HDMECs reduced cell migration and basal cell membrane permeability, and increased capillary-like tube formation. Based on these results, we believe Reelin is involved in vasculogenesis and angiogenesis. This prompted us to analyze expression of angiogenic transcripts by qPCR array in negative control and RELN knockdown (KD) HDMECs. Our results indicated significant upregulation of ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs), and significant changes in expression of other angiogenesis-related transcripts.
Study Objective: This study aimed to validate the angiogenesis array data by individual TaqMan qPCR analysis of mRNA expression of the significantly altered transcripts. Also, we aimed to analyze the encoded protein expression of these transcripts in negative control and RELN KD cells. We hypothesized that significant alterations in expression of ADAMTS1 and other angiogenic transcripts leads to changes in their protein expression in our RELN KD cells and mediates Reelin signaling in human vascular endothelial cells.
Methods: To validate our angiogenesis qPCR array data, we utilized HDMECs as an in vitro system to examine mRNA and protein expression. Gene-specific siRNAs were used to target and knockdown endogenous expression of RELN in HDMECs. Total RNA was isolated from negative control and RELN KD HDMECs and it was analyzed by qPCR with TaqMan probes for RELN, ADAMTS1, GAPDH, 18SRNA and other angiogenic transcripts. Also, protein expression of the encoded transcripts was analyzed by immunofluorescence and western blot in negative control and RELN KD HDMECs.
Results: RELN KD HDMECs exhibited a >90% reduction in Reelin protein expression compared to negative control HDMECs. ADAMTS1 mRNA was significantly upregulated in RELN KD cells while expression of other angiogenic transcripts were also altered in these cells. We demonstrated by immunofluorescence that ADAMTS1 is localized to the nucleus, and it is detected in HDMEC lysates by western blot analysis. In addition, ADAMTS1 protein expression differed between negative control and RELN KD HDMECs.
Conclusions: We conclude that ADAMTS1 mRNA is upregulated in RELN KD HDMECs versus negative control cells. Knockdown of RELN in HDMECS altered cell migration, membrane permeability, capillary-like tube formation and expression of angiogenic transcripts. These findings provide key evidence that ADAMTS1 and other angiogenic transcripts may be novel mediators of Reelin signaling in human vascular endothelial cells.
Comments
Winner of PCOM Philadelphia David Miller, DO ‘60 Memorial Research Day Best in Show Award
David Miller, DO ‘60 Memorial Research Day Best in Show Award