Effects of diet, BMP‐2 treatment, and femoral skeletal injury on endothelial cells derived from the ipsilateral and contralateral limbs

Abstract

Type 2 diabetes (T2D) results in physiological and structural changes in bone, contributing to poor fracture healing. T2D compromises microvascular performance, which can negatively impact bone regeneration as angiogenesis is required for new bone formation. We examined the effects of bone morphogenetic protein‐2 (BMP‐2) administered locally at the time of femoral segmental bone defect (SBD) surgery, and its angiogenic impacts on endothelial cells (ECs) isolated from the ipsilateral or contralateral tibia in T2D mice. Male C57BL/6 mice were fed either a low‐fat diet (LFD) or high‐fat diet (HFD) starting at 8 weeks. After 12 weeks, the T2D phenotype in HFD mice was confirmed via glucose and insulin tolerance testing and echoMRI, and all mice underwent SBD surgery. Mice were treated with BMP‐2 (5 µg) or saline at the time of surgery. Three weeks postsurgery, bone marrow ECs were isolated from ipsilateral and contralateral tibias, and proliferation, angiogenic potential, and gene expression of the cells was analyzed. BMP‐2 treatment increased EC proliferation by two fold compared with saline in LFD contralateral tibia ECs, but no changes were seen in surgical tibia EC proliferation. BMP‐2 treatment enhanced vessel‐like structure formation in HFD mice whereas, the opposite was observed in LFD mice. Still, in BMP‐2 treated LFD mice, ipsilateral tibia ECs increased expression of CD31, FLT‐1, ANGPT1, and ANGPT2. These data suggest that the modulating effects of T2D and BMP‐2 on the microenvironment of bone marrow ECs may differentially influence angiogenic properties at the fractured limb versus the contralateral limb.

Characterization and assessment of lung and bone marrow derived endothelial cells and their bone regenerative potential

“…Neonatal LECs are isolated specifically with CD31, a requisite EC marker, making their population homogenous, while neonatal BMECs are comprised of a heterogeneous population that may be composed of stalk and tip cells, ECs, parenchymal cells, and endothelial cell-like cells ( 33 ). These differences may influence the cells performance in the assays tested.…”

“…All tissue isolation was performed in a sterile environment. LECs and BMECs were isolated from neonatal mice (2-6 days of age) as described previously ( 4 , 32 , 33 ).…”

“…cDNA was prepared using the SuperScript ® III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, Carlsbad, CA). Quantitative PCR was performed using FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics GmbH, Mannheim, Germany) as previously described ( 33 ). The following genes were analyzed: CD31 Antigen (CD31), Vascular Endothelial Growth Factor (VEGF), Fms Related Tyrosine Kinase 1 (Flk-1), Fms Related Receptor Tyrosine Kinase 1 (Flt-1), Angiopoietin 1 (ANGPT1), and Angiopoietin 2 (ANGPT2).…”

The Effects of SRT1720 Treatment on Endothelial Cells Derived from the Lung and Bone Marrow of Young and Aged, Male and Female Mice

“…Compared to LECs, BMECs exhibited significantly more angiogenic mRNA expression differences following treatment with SRT1720. We speculate that this may occur as LECs are isolated as a homogenous CD31+ population whereas BMECs are isolated as a heterogeneous population [ 45 , 46 , 47 , 48 , 49 ] that is comprised of mature and immature EC cells, stalk and tip cells, and perhaps EC-like cells that assist in EC function [ 11 ]. Exploring and comparing the gene profiles between these subpopulations of ECs remains to be investigated but is important to better understand how drugs would impact multiple cell types.…”

“…BMECs remained in this culture for seven days before being used for experiments. We have previously characterized BMECs isolated from this procedure by a number of criteria, including the examination of Tie2 expression (as evidenced in BMECs isolated from mice in which tdTomato is expressed under the control of the Tie2 promoter following activation with tamoxifen), CD31 immunocytochemical staining, and flow cytometric analysis (CD45- Ter119- CD31+ CD105+ cells) [ 47 , 48 , 49 ].…”