Medial Hypoxia and Adventitial Vasa Vasorum Remodeling in Human Ascending Aortic Aneurysm

Human ascending aortic aneurysms characteristically exhibit cystic medial degeneration of the aortic wall encompassing elastin degeneration, proteoglycan accumulation and smooth muscle cell loss. Most studies have focused on the aortic media and there is a limited understanding of the importance of the adventitial layer in the setting of human aneurysmal disease. We recently demonstrated that the adventitial ECM contains key angiogenic factors that are downregulated in aneurysmal aortic specimens. In this study, we investigated the adventitial microvascular network (vasa vasorum) of aneurysmal aortic specimens of different etiology and hypothesized that the vasa vasorum is disrupted in patients with ascending aortic aneurysm. Morphometric analyses of hematoxylin and eosin-stained human aortic cross-sections revealed evidence of vasa vasorum remodeling in aneurysmal specimens, including reduced density of vessels, increased lumen area and thickening of smooth muscle actin-positive layers. These alterations were inconsistently observed in specimens of bicuspid aortic valve (BAV)-associated aortopathy, while vasa vasorum remodeling was typically observed in aneurysms arising in patients with the morphologically normal tricuspid aortic valve (TAV). Gene expression of hypoxia-inducible factor 1α and its downstream targets, metallothionein 1A and the pro-angiogenic factor vascular endothelial growth factor, were down-regulated in the adventitia of aneurysmal specimens when compared with non-aneurysmal specimens, while the level of the anti-angiogenic factor thrombospondin-1 was elevated. Immunodetection of glucose transporter 1 (GLUT1), a marker of chronic tissue hypoxia, was minimal in non-aneurysmal medial specimens, and locally accumulated within regions of elastin degeneration, particularly in TAV-associated aneurysms. Quantification of GLUT1 revealed elevated levels in the aortic media of TAV-associated aneurysms when compared to non-aneurysmal counterparts. We detected evidence of chronic inflammation as infiltration of lymphoplasmacytic cells in aneurysmal specimens, with a higher prevalence of lymphoplasmacytic infiltrates in aneurysmal specimens from patients with TAV compared to that of patients with BAV. These data highlight differences in vasa vasorum remodeling and associated medial chronic hypoxia markers between aneurysms of different etiology. These aberrations could contribute to malnourishment of the aortic media and could conceivably participate in the pathogenesis of thoracic aortic aneurysm

Classification and Functional Characterization of Vasa Vasorum-Associated Perivascular Progenitor Cells in Human Aorta

In the microcirculation, pericytes are believed to function as mesenchymal stromal cells (MSCs). We hypothesized that the vasa vasorum harbor progenitor cells within the adventitia of human aorta. Pericytes, endothelial progenitor cells, and other cell subpopulations were detected among freshly isolated adventitial cells using flow cytometry. Purified cultured pericytes were enriched for the MSC markers CD105 and CD73 and depleted of the endothelial markers von Willebrand factor and CD31. Cultured pericytes were capable of smooth muscle lineage progression including inducible expression of smooth muscle myosin heavy chain, calponin, and α-smooth muscle actin, and adopted a spindle shape. Pericytes formed spheroids when cultured on Matrigel substrates and peripherally localized with branching endothelial cells in vitro. Our results indicate that the vasa vasorum form a progenitor cell niche distinct from other previously described progenitor populations in human adventitia. These findings could have important implications for understanding the complex pathophysiology of human aortic disease

Mechanism of aortic medial matrix remodeling is distinct in patients with bicuspid aortic valve

ObjectivesPatients with bicuspid aortic valves (BAV) are predisposed to developing ascending thoracic aortic aneurysms (TAA) at an earlier age than patients who develop degenerative TAAs and have a tricuspid aortic valve (TAV). The hypothesis tested is that BAV-associated aortopathy is mediated by a mechanism of matrix remodeling that is distinct from that seen in TAAs of patients with tricuspid aortic valves.MethodsAortic specimens were collected during ascending aortic replacement, aortic valve replacement, and heart transplants from nonaneurysmal (NA) donors and recipients. Matrix architecture of the aortic media was assessed qualitatively using multiphoton microscopy followed by quantification of collagen and elastin fiber orientation. α-Elastin was determined and matrix maturity was assessed by quantifying immature and mature collagen and lysyl oxidase (Lox) expression and activity in aortic specimens. Matrix metalloproteinase-2/9 activity was quantified in aortic smooth muscle cells.ResultsElastin and collagen fibers were more highly aligned in BAV-NA and BAV-TAA cases than in TAV-TAA cases, whereas TAV-TAA cases were more disorganized than TAV-NA cases. α-Elastin content was unchanged. Immature collagen was reduced in BAV-NA and BAV-TAA cases when compared with TAV-NA and TAV-TAA cases. Mature collagen was elevated in TAV-TAA cases compared with TAV-NA and BAV-TAA cases. There was a trend toward elevated Lox gene expression and activity and matrix metalloproteinase-2/9 activity for TAV-TAA, BAV-NA, and BAV-TAA specimens.ConclusionsThe highly aligned matrix architecture in patients with BAVs indicates that wall remodeling is distinct from TAV-TAA. Altered matrix architecture and reduced collagen maturity suggest that the effector molecules mediating the remodeling of TAAs are different in BAV and TAV cases

Effect of Phosphatidyl Inositol 3-Kinase, Extracellular Signal-Regulated Kinases 1/2, and p38 Mitogen-Activated Protein Kinase Inhibition on Osteogenic Differentiation of Muscle-Derived Stem Cells

Skeletal muscle-derived stem cells (MDSCs) can undergo osteogenesis when treated with bone morphogenetic proteins (BMPs), making them a potential cell source for bone tissue engineering. The signaling pathways that regulate BMP4-induced osteogenesis in MDSCs are not well understood, although they may provide a means to better regulate differentiation during bone regeneration. The objective of this study was to characterize the signaling pathways involved in the BMP4-induced osteogenesis of MDSCs. Cells were treated with BMP4 and specific inhibitors to the extracellular signal-regulated kinases 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and phosphatidyl inositol 3-kinase (PI3K) pathways (PD98059, SB203580, and Ly294002, respectively). Cellular proliferation, expression of osteoblast-related genes, alkaline phosphatase (ALP) activity, and tissue mineralization were measured to determine the role of each pathway in the osteogenic differentiation of MDSCs. Inhibition of the ERK1/2 pathway increased ALP activity and mineralization, whereas inhibition of the p38 MAPK pathway decreased osteogenesis, suggesting opposing roles of these pathways in the BMP4-induced osteogenesis of MDSCs. Inhibition of the PI3K pathway significantly increased mineralization by MDSCs. These findings highlight the involvement of the ERK1/2, p38 MAPK, and PI3K pathways in opposing capacities in MDSC differentiation and warrant further investigation, as it may identify novel therapeutic targets for the development of stem cell-based therapies for bone tissue engineering