Qingbo Xu

Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice

Recent evidence indicates that vascular progenitor cells may be the source of smooth muscle cells (SMCs) that accumulate in atherosclerotic lesions, but the origin of these progenitor cells is unknown. To explore the possibility of vascular progenitor cells existing in adults, a variety of tissues from ApoE-deficient mice were extensively examined. Immunohistochemical staining revealed that the adventitia in aortic roots harbored large numbers of cells having stem cell markers, e.g., Sca-1(+) (21%), c-kit(+) (9%), CD34(+) (15%), and Flk1(+) cells (4%), but not SSEA-1(+) embryonic stem cells. Explanted cultures of adventitial tissues using stem cell medium displayed a heterogeneous outgrowth, for example, islands of round-shaped cells surrounded by fibroblast-like cell monolayers. Isolated Sca-1(+) cells were able to differentiate into SMCs in response to PDGF-BB stimulation in vitro. When Sca-1(+) cells carrying the LacZ gene were transferred to the adventitial side of vein grafts in ApoE-deficient mice, beta-gal(+) cells were found in atherosclerotic lesions of the intima, and these cells enhanced the development of the lesions. Thus, a large population of vascular progenitor cells existing in the adventitia can differentiate into SMCs that contribute to atherosclerosis. Our findings indicate that ex vivo expansion of these progenitor cells may have implications for cellular, genetic, and tissue engineering approaches to vascular disease.

Is atherosclerosis an immunologically mediated disease

In contrast to general beliefs, recent data from different laboratories have provided evidence that the first stages of atherosclerosis are of an inflammatory nature. Here, Georg Wick and colleagues suggest that an autoimmune reaction against heat shock protein 60 (Hsp60), expressed by endothelial cells in areas that are subject to increased haemodynamic stress, is the initiating event in atherogenesis. Humoral and T-cell-mediated immune responses against Hsp60 have both been demonstrated early in disease. This inflammatory stage, which is reversible and has even been found in children, may progress into fully developed atherosclerotic lesions, displaying all the classical pathohistological and functional consequences, if additional risk factors such as high blood cholesterol levels, smoking and obesity, are present.

Vascular repair by endothelial progenitor cells

Accumulating evidence indicates the impact of endothelial progenitor cells (EPCs) in vascular repair. In patients, the number of EPCs is negatively correlated with the severity of atherosclerosis. In various animal models, transplantation of bone marrow-derived progenitor cells could sufficiently rescue organ function and enhance vascular repair and tissue regeneration. Increase in the number of circulating progenitors, induced by cell transfusion or enhanced mobilization, can also enhance restoration and integrity of the endothelial lining, suppress neointimal formation, and increase blood flow to ischaemic sites. However, the beneficial outcome of EPC infusion very much depends on the growth and differentiation factors within the tissue, cell-to-cell interactions, and the degree of injury. As highlighted by several studies, EPCs derive from different sources including bone marrow and non-bone marrow organs such as the spleen, the functional repair properties of which may vary with the maturation state of the cell. Thus, understanding the molecular mechanisms involved in EPC-repairing processes is essential. In the present review we focus on the role of EPCs in vascular diseases, and we provide an update on the mechanisms of EPC mobilization, homing, and differentiation.

Serum Soluble Heat Shock Protein 60 Is Elevated in Subjects With Atherosclerosis in a General Population

BACKGROUND Work from our laboratory has proven that increased titers of anti-heat shock protein 60 (HSP60) antibodies are associated with atherosclerosis and that HSP60-reactive T-cells are present in atherosclerotic lesions. Recent studies from others demonstrated that HSP60 directly activates endothelial cells and macrophages. METHODS AND RESULTS To explore the possibility that HSP60 exists in the circulation, where it could exert its functions, we performed a population-based study with 826 subjects aged 40 to 79 years. The following items were measured in all participants: serum soluble HSP60 (sHSP60); anti-Escherichia coli lipopolysaccharide; anti-HSP65, anti-Chlamydia and anti-Helicobacter pylori antibodies; and a variety of acute phase reactants (C-reactive protein, alpha(1)-antitrypsin, and ceruloplasmin) and markers of systemic inflammation. Carotid atherosclerosis was assessed twice (1990 and 1995), and 15 other risk factors were evaluated. Our data show that levels of sHSP60 were significantly elevated in subjects with prevalent/incident carotid atherosclerosis and that these levels were correlated with common carotid artery intima/media thickness. Multiple logistic regression analysis documented these associations as independent of age, sex, and other risk factors. Interestingly, sHSP60 was also correlated with anti-lipopolysaccharide, anti-Chlamydia and anti-HSP60 antibodies, various markers of inflammation, and the presence of chronic infections. The risk of atherosclerosis associated with high sHSP60 levels was amplified when subjects had clinical and/or laboratory evidence of chronic infections. CONCLUSIONS Our data provide the first evidence of a strong correlation between sHSP60 and atherosclerosis, suggesting that sHSP60 may play important roles in activating vascular cells and the immune system during the development of atherosclerosis.

Chronic Infections and the Risk of Carotid Atherosclerosis

Background—Chronic infections have been implicated in the pathogenesis of atherosclerosis, yet from an epidemiological perspective, this concept remains controversial. Methods and Results—The Bruneck Study is a prospective population-based survey on the pathogenesis of atherosclerosis. In 826 men and women 40 to 79 years old (1990 baseline), 5-year changes in carotid atherosclerosis were thoroughly assessed by high-resolution duplex scanning. The presence of chronic respiratory, urinary tract, dental, and other infections was ascertained by standard diagnostic criteria. Chronic infections amplified the risk of atherosclerosis development in the carotid arteries. The association was most pronounced in subjects free of carotid atherosclerosis at baseline (age-/sex-adjusted odds ratio [95% CI] for any chronic infection versus none, 4.08 [2.42 to 6.85]; P<0.0001) and applied to all types of chronic (bacterial) infections. It remained independently significant after adjustment for classic vascular risk attribu...

Exacerbated vein graft arteriosclerosis in protein kinase Cδ–null mice

Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCdelta knockout mice and performed vein bypass grafts on these animals. PKCdelta(-/-) mice developed normally and were fertile. Vein segments from PKCdelta(-/-) mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCdelta(+/+) vein grafts. Arteriosclerotic lesions in PKCdelta(-/-) mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCdelta(-/-) mice. SMCs derived from PKCdelta(-/-) aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCdelta(-/-) relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCdelta maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.

Oxidized Phospholipids, Lipoprotein(a), Lipoprotein-Associated Phospholipase A2 Activity, and 10-Year Cardiovascular Outcomes Prospective Results From the Bruneck Study

Background—Oxidized phospholipids (OxPL) circulate on apolipoprotein B-100 particles (OxPL/apoB), and primarily on Lp(a) lipoprotein (a) [Lp(a)]. The relationship of OxPL/apoB levels to future cardiovascular events is not known. Methods and Results—The Bruneck study is a prospective population-based survey of 40- to 79-year-old men and women recruited in 1990. Plasma levels of OxPL/apoB and lipoprotein (a) [Lp(a)] were measured in 765 subjects in 1995 and incident cardiovascular disease (CVD), defined as cardiovascular death, myocardial infarction, stroke, and transient ischemic attack, was assessed from 1995 to 2005. During the follow-up period, 82 subjects developed CVD. In multivariable analysis, which included traditional risk factors, high sensitivity C-reactive protein (hsCRP), and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, subjects in the highest tertile of OxPL/apoB had a significantly higher risk of cardiovascular events than those in the lowest tertile (hazard ratio[95% CI] 2.4[1.3 to 4.3], P=0.004). The strength of the association between OxPL/apoB and CVD risk was amplified with increasing Lp-PLA2 activity (P=0.018 for interaction). Moreover, OxPL/apoB levels predicted future cardiovascular events beyond the information provided by the Framingham Risk Score (FRS). The effects of OxPL/apoB and Lp(a) were not independent of each other but they were independent of all other measured risk factors. Conclusions—This study demonstrates that OxPL/apoB levels predict 10-year CVD event rates independently of traditional risk factors, hsCRP, and FRS. Increasing Lp-PLA2 activity further amplifies the risk of CVD mediated by OxPL/apoB.

HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell–derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1–PI3K–Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor–induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell–derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1–PI3K–Akt–HDAC3–p53–p21 pathway is crucial in such a process.

Activation of PDGF receptor α in vascular smooth muscle cells by mechanical stress

Hypertension increases mechanical force on the arterial wall by as much as 30%, resulting in marked alterations in signal transductions and gene expression in vascular smooth muscle cells (VSMCs) that contribute to matrix protein synthesis, cell proliferation, and differentiation. How the mechanical stimuli are converted into a biological signal in cells has yet to be studied. We investigated the role of both cyclic strain and shear stresses in initiating the cellular signaling on cultured VSMCs and found that mechanical forces evoked activation of mitogen‐activated protein kinases, followed by enhanced DNA binding activity of transcription factor AP‐1. Physical forces rapidly induced phosphorylation of platelet‐derived growth factor receptor (PDGFR) α, an activated state. When GRB2, an adapter protein, was immunoprecipitated from treated VSMCs followed by Western blot analysis with anti‐phosphotyrosine, ‐PDGFRα, and ‐GRB2 antibodies, respectively, phosphotyrosine positive staining was observed on PDGFRα bands of the same blot in stretch‐stressed VSMCs, supporting the mechanical stress‐induced activation of PDGFRα. Conditioned medium from stretch‐stressed VSMCs did not result in PDGFRα phosphorylation, and antibodies binding to all forms of PDGFs did not block stress‐induced PDGFRα activation. Thus, mechanical stresses may directly perturb the cell surface or alter receptor conformation, thereby initiating signaling pathways normally used by growth factors.—Hu, Y., Böck, Güunther, Wick, G., Xu, Q., Activation of PDGF receptor α in vascular smooth muscle cells by mechanical stress. FASEB J. 12, 1135–1142 (1998)

Endothelial Replacement and Angiogenesis in Arteriosclerotic Lesions of Allografts Are Contributed by Circulating Progenitor Cells

Background—Endothelial regeneration and angiogenesis in the intima of the arterial wall are key events in the pathogenesis of transplantation arteriosclerosis. The traditional hypothesis that damaged endothelial cells are replaced by remaining cells of the donor vessel has been challenged by recent observations, but the cell origins of large arteries and microvessels are still not well established. Methods and Results—Aortic segments were allografted between Balb/c and TIE2-LacZ (C57BL/6) mice expressing &bgr;-galactosidase (gal) in endothelial cells. &bgr;-gal+ cells in TIE2-LacZ vessels grafted to Balb/c mice completely disappeared, whereas the positive cells found in Balb/c aorta allografted into TIE2-LacZ mice 4 weeks after surgery indicated a host origin. En face analysis of allograft vessels displayed a unique distribution of &bgr;-gal+ cells on the surface at 3 days, 1 week, and 4 weeks. Interestingly, 35±19% &bgr;-gal+ cells were found in arterial segments allografted into chimeric mice with TIE2-LacZ bone marrows. Furthermore, endothelial cells of microvessels within allografts had a &bgr;-gal+ staining in the media at 1 week and in the neointimal lesions and adventitia at 4 weeks. Allograft studies in chimeric mice demonstrated that &bgr;-gal+ cells of microvessels in transplant arteriosclerosis were derived from bone marrow progenitors. Conclusions—We provide strong evidence that endothelial cells of neointimal lesions in allografts are derived from circulating progenitor cells and that bone marrow–derived progenitors are responsible for angiogenesis of the allograft, that is, the formation of microvessels in transplant arteriosclerosis.