School of Medicine and Health Sciences Poster Presentations

Title

Transplanting P21KO EPCs May Improve Vascularization in Diabetes Related PVD

Poster Number

131

Document Type

Poster

Publication Date

3-2016

Abstract

Background: One of the major secondary effects of diabetes is peripheral vascular disease (PVD). Unfortunately, 60% of non –traumatic lower limb amputation is related to PVD and is a consequence of diabetes. Therefore, it is important to treat PVD aggressively to prevent mobility handicap for subjects with diabetes. Studies indicate that delivery of endothelial progenitor cells (EPCs) improve vascularization in mouse model. However, major concern is to ensure survival of these cells in hyper glycemic condition. Previous study from our laboratory showed improved survival and better collateral vessel formation when p53KO EPCs were delivered post femoral artery occlusion in diabetic mouse models. In this study, we wanted to investigate whether transplantation of EPCs from P21 knock out (P21KO) mice at the site of femoral artery occlusion will improve regeneration and new collateral vessel formation. p21 is a downstream protein in the apoptosis cascade and therefore silencing of p21 rather than p53 may have less off-target effects. While investigating apoptosis resistant endothelial progenitor stem cells (EPCs) in diabetic mouse models we enquired into effect of hyperglycemia on mature human endothelial cells (HUVEC) in a time-course dependant manner.

Method: We exposed mature HUVEC (Human Umbilical Vein Endothelial Cell) and EPCs in normal (5.5mM) and high glucose (20mM) and carried out gene expression studies and assessed their respiration rate at different time points. We transplanted EPCs from p21KO mice in streptozotocin (STZ) induced diabetic mice (type 1 diabetes model) post occlusion of the right femoral artery. We measured blood flow at days 3, 7, 10, 14 and 21 after surgery and did gene expression study and stain quadriceps with CD31 antibody to determine vascularization.

Results: We observed reduction in respiration rate of HUVEC only after 28 days of exposure to hyperglycemic condition which was associated with upregulation of P53, P21 and inflammatory markers after 28 days of exposer in hyperglycemic condition in HUVEC cells. Whereas, upregulation of P53 and P21 was noted within 48 hours of hyperglycemic condition exposure in EPCs.

For our in vivo studies using p21KO, wild type (WT) and saline in STZ induced diabetic mice, we observed enhanced vascularization on the hind limb after surgery and delivery of p21 KO EPCs, compared to WT and saline. Simultaneously, we also observed upregulation of endothelial markers like PECAM1, KDR, vWF and eNOS in hindlimb muscles that received p21 KO EPCs. Staining with CD31 is also showed improved vascularization in p21KO EPC injected mouse quadriceps.

Conclusion: These studies suggest that apoptosis of cells particularly stem cells plays a major role and prevention of cellular apoptosis may prove to be an important cell based therapy for diabetic PVD and limb salvage.

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Presented at: GW Research Days 2016

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Transplanting P21KO EPCs May Improve Vascularization in Diabetes Related PVD

Background: One of the major secondary effects of diabetes is peripheral vascular disease (PVD). Unfortunately, 60% of non –traumatic lower limb amputation is related to PVD and is a consequence of diabetes. Therefore, it is important to treat PVD aggressively to prevent mobility handicap for subjects with diabetes. Studies indicate that delivery of endothelial progenitor cells (EPCs) improve vascularization in mouse model. However, major concern is to ensure survival of these cells in hyper glycemic condition. Previous study from our laboratory showed improved survival and better collateral vessel formation when p53KO EPCs were delivered post femoral artery occlusion in diabetic mouse models. In this study, we wanted to investigate whether transplantation of EPCs from P21 knock out (P21KO) mice at the site of femoral artery occlusion will improve regeneration and new collateral vessel formation. p21 is a downstream protein in the apoptosis cascade and therefore silencing of p21 rather than p53 may have less off-target effects. While investigating apoptosis resistant endothelial progenitor stem cells (EPCs) in diabetic mouse models we enquired into effect of hyperglycemia on mature human endothelial cells (HUVEC) in a time-course dependant manner.

Method: We exposed mature HUVEC (Human Umbilical Vein Endothelial Cell) and EPCs in normal (5.5mM) and high glucose (20mM) and carried out gene expression studies and assessed their respiration rate at different time points. We transplanted EPCs from p21KO mice in streptozotocin (STZ) induced diabetic mice (type 1 diabetes model) post occlusion of the right femoral artery. We measured blood flow at days 3, 7, 10, 14 and 21 after surgery and did gene expression study and stain quadriceps with CD31 antibody to determine vascularization.

Results: We observed reduction in respiration rate of HUVEC only after 28 days of exposure to hyperglycemic condition which was associated with upregulation of P53, P21 and inflammatory markers after 28 days of exposer in hyperglycemic condition in HUVEC cells. Whereas, upregulation of P53 and P21 was noted within 48 hours of hyperglycemic condition exposure in EPCs.

For our in vivo studies using p21KO, wild type (WT) and saline in STZ induced diabetic mice, we observed enhanced vascularization on the hind limb after surgery and delivery of p21 KO EPCs, compared to WT and saline. Simultaneously, we also observed upregulation of endothelial markers like PECAM1, KDR, vWF and eNOS in hindlimb muscles that received p21 KO EPCs. Staining with CD31 is also showed improved vascularization in p21KO EPC injected mouse quadriceps.

Conclusion: These studies suggest that apoptosis of cells particularly stem cells plays a major role and prevention of cellular apoptosis may prove to be an important cell based therapy for diabetic PVD and limb salvage.