ACE2 and AT4R are present in diseased human blood vessels

A growing body of evidence suggests that the angiotensin II fragments, Ang(1-7) and Ang(3-8), have a vasoactive role, however ACE2, the enzyme that produces Ang(1-7), or AT4R, the receptor that binds Ang (3-8), have yet been simultaneously localised in both normal and diseased human conduit blood vessels. We sought to determine the immunohistochemical distribution of ACE2 and the AT4R in human internal mammary and radial arteries from patients undergoing coronary artery bypass surgery. We found that ACE2 positive cells were abundant in both normal and diseased vessels, being present in neo-intima and in media. ACE2 positive immunoreactivity was not present in the endothelial layer of the conduit vessels, but was clearly evident in small newly formed angiogenic vessels as well as the vaso vasorum. Endothelial AT4R immunoreactivity were rarely observed in either normal and diseased arteries, but AT4R positive cells were observed adjacent to the internal elastic lamine in the internal mammary artery, in the neo-intima of radial arteries, as well as in the media of both internal mammary artery and radial artery. AT4R was abundant in vaso vasorum and within small angiogenic vessels. Both AT4R and ACE2 co-localised with smooth muscle cell alpha actin. This study identifies smooth muscle cell alpha actin positive ACE2 and AT4R in human blood vessels as well as in angiogenic vessels, indicating a possible role for these enzymes in pathological disease.     

Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG

The ends of human chromosomes are protected from the degradation associated with cell division by 15-20 kb long segments of hexameric repeats of 5'-TTAGGG-3' termed telomeres. In normal cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; however, most cancer cells bypass this lifespan restriction through the expression of telomerase. hTERT, the catalytic subunit essential for the proper function of telomerase, has been shown to be expressed in approximately 90% of all cancers. In this study we investigated the hTERT inhibiting effects of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea catechins, in MCF-7 breast cancers cells and HL60 promyelocytic leukemia cells. Exposure to EGCG reduced cellular proliferation and induced apoptosis in both MCF-7 and HL60 cells in vitro, although hTERT mRNA expression was decreased only in MCF-7 cells when treated with EGCG. Furthermore, down-regulation of hTERT gene expression in MCF-7 cells appeared to be largely due to epigenetic alterations. Treatment of MCF-7 cells with EGCG resulted in a time-dependent decrease in hTERT promoter methylation and ablated histone H3 Lys9 acetylation. In conjunction with demethylation, further analysis showed an increase in hTERT repressor E2F-1 binding at the promoter. From these findings, we propose that EGCG is effective in causing cell death in both MCF-7 and HL60 cancer cell lines and may work through different pathways involving both anti-oxidant effects and epigenetic modulation.     

Increased Expression of Phosphorylated Polo-Like Kinase 1 and Histone in Bypass Vein Graft and Coronary Arteries following Angioplasty

Interventional procedures, including percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass surgery (CABG) to re-vascularize occluded coronary arteries, injure the vascular wall and cause endothelial denudation and medial vascular smooth muscle cell (VSMCs) metaplasia. Proliferation of the phenotypically altered SMCs is the key event in the pathogenesis of intimal hyperplasia (IH). Several kinases and phosphatases regulate cell cycle in SMC proliferation. It is our hypothesis that increased expression and activity of polo-like kinase-1 (PLK1) in SMCs, following PTCA and CABG, contributes to greater SMC proliferation in the injured than uninjured blood vessels. Using immunofluorescence (IF), we assessed the expression of PLK1 and phosphorylated-PLK1 (pPLK1) in post-PTCA coronary arteries, and superficial epigastric vein grafts (SEV) and compared it with those in the corresponding uninjured vessels. We also compared the expressions of mitotic marker phospho-histone, synthetic-SMC marker, contractile SMC marker, IFN-γ and phosphorylated STAT-3 in the post-PTCA arteries, SEV-grafts, and the uninjured vessels. Immunostaining demonstrated an increase in the number of cells expressing PLK1 and pPLK1 in the neointima of post PTCA-coronary arteries and SEV-grafts compared to their uninjured counterparts. VSMCs in the neointima showed an increased expression of phospho-histone, synthetic and contractile SMC markers, IFN-γ and phosphorylated STAT-3. However, VSMCs of uninjured coronaries and SEV had no significant expression of the aforementioned proteins. These data suggest that PLK1 might play a critical role in VSMC mitosis in hyperplastic intima of the injured vessels. Thus, novel therapies to inhibit PLK1 could be developed to inhibit the mitogenesis of VSMCs and control neointimal hyperplasia.     

Arterial territory-specific phosphorylated retinoblastoma protein species and CDK2 promote differences in the vascular smooth muscle cell response to mitogens

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.     

Microvessel precursor smooth muscle cells express head-inserted smooth muscle myosin heavy chain (SM-B) isoform in hyperoxic

The present study analyzes smooth muscle myosin heavy chain (SMMHC) expression as lung microvascular precursor smooth muscle cells (PSMCs), cells derived from fibroblasts and intermediate cells (immature SMCs), acquire a smooth muscle phenotype in anin vivo model of pulmonary hypertension (PH). Because of the unique contractile properties of the SMMHC isoform SM-B, we analyzed its expression in the microvessels (<100 μm diameter) and in larger vessels (100–700 μm) quantitatualy by the labeled [strept]avidin-biotin technique (day 1–28), and related this to cell phenotype by transmission microscopy and protein A-gold labeling (at day 28). Airway SMCs of the normal and hypertensive lung uniformly expressed SM-B whereas vascular SMC expression was heterogeneous. Thus, in some large arteries (and veins) SMCs contained cells expressing SM-B while in others all the cells were immunonegative. Microvascular cells expressing SM-B (arteries and veins) were rare in normal lung and numerous in PH, increasing as wall muscle developed in smaller segments with time. As in large vessels, some microvessels had immunopositive cells and others only negative ones. Ultrastructural analysis confirmed that the SMCs of bronchial vessels, and the septal SMCs adjoining alveolar ducts, contained dense filament arrays decorated with SM-B. While the PSMC processes of the normal lung contained sparse filaments decorated with SM-B, these cells expressed dense filament arrays in PH. Fibroblasts migrating to align around the microvessels also expressed SM-B but in the absence of a filament network. For the first time,we demonstrate in vivo that newly developed microvascular PSMCs express the SMMHC SM-B isoform in PH. Received: 9 April 1998 / Accepted: 9 September 1998     

Telomerase maintains telomere structure in normal human cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.     

Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts

The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process.     

Extension of human cell lifespan by nicotinamide phosphoribosyltransferase

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated beta-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.     

Stabilization of telomere length and karyotypic stability are directly correlated with the level of hTERT gene expression in primary fibroblasts

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase (hTERT) gene in sheep fibroblasts reconstitutes telomerase activity and extends their lifespan. However, telomere length is not maintained in all cell lines, even though in vitro telomerase activity is restored in all of them. Cell lines expressing higher levels of hTERT mRNA do not exhibit telomere erosion or genomic instability. By contrast, fibroblasts expressing lower levels of hTERT do exhibit telomere shortening, although the telomeres eventually stabilize at a shorter length. The shorter telomere lengths and the extent of karyotypic abnormalities are both functions of hTERT expression level. We conclude that telomerase activity is required to bypass senescence but is not sufficient to prevent telomere erosion and genomic instability at lower levels of expression.     

High-salt diet advances molecular circadian rhythms in mouse peripheral tissues

Most human somatic cells contain no or very low levels of telomerase. The over-expression of the catalytic subunit (hTERT) of human telomerase is a common method to generate cells with a greatly prolonged lifespan. These cells serve as models for cells that are either difficult to cultivate or have a limited lifespan in vitro. In addition, hTERT over-expressing cells are thought to be a useful resource for tissue engineering and regenerative medicine.While tumour suppressors and cell cycle checkpoints are maintained for an extended period in most hTERT over-expressing cells we found that there is a gradual change in gene expression over a range of 130 population doublings (PD) for the majority of genes analysed. Seven genes were significantly down-regulated with increasing population doublings (PDs), while only two were up-regulated. One gene, stanniocalcin 2, was highly expressed in parental fibroblasts but completely diminished as a consequence of hTERT transgene expression.These data demonstrate that in hTERT over-expressing cells two different types of expression changes occur: one can be directly associated with hTERT transgene expression itself, while others might occur more gradual and with varying kinetics. These changes should be taken into account when these cells are used as functional models or for regenerative purposes.     

SIRT1 markedly extends replicative lifespan if the NAD salvage pathway is enhanced

Sir2 mediates lifespan extension in lower eukaryotes but whether its mammalian homolog, sirtuin 1, silent mating type information regulation 2 homolog (SIRT1), is a longevity protein is controversial. We stably introduced the SIRT1 gene into human vascular smooth muscle cells (SMCs) and observed minimal extension of replicative lifespan. However, SIRT1 activity was found to be exquisitely dependent on nicotinamide phosphoribosyltransferase (Nampt) activity. Moreover, overexpression of Nampt converted SIRT1-overexpressing SMCs to senescence-resistant cells together with heightened SIRT1 activity, suppressed p21, and strikingly lengthened replicative lifespan. Thus, SIRT1 can markedly postpone SMC senescence, but this requires overcoming an otherwise vulnerable nicotinamide adenine dinucleotide salvage reaction in aging SMCs.     

Fibroblast growth factor 9 delivery during angiogenesis produces durable, vasoresponsive microvessels wrapped by smooth muscle cells

The therapeutic potential of angiogenic growth factors has not been realized. This may be because formation of endothelial sprouts is not followed by their muscularization into vasoreactive arteries. Using microarray expression analysis, we discovered that fibroblast growth factor 9 (FGF9) was highly upregulated as human vascular smooth muscle cells (SMCs) assemble into layered cords. FGF9 was not angiogenic when mixed with tissue implants or delivered to the ischemic mouse hind limb, but instead orchestrated wrapping of SMCs around neovessels. SMC wrapping in implants was driven by sonic hedgehog-mediated upregulation of PDGFRβ. Computed tomography microangiography and intravital microscopy revealed that microvessels formed in the presence of FGF9 had enhanced capacity to receive flow and were vasoreactive. Moreover, the vessels persisted beyond 1 year, remodeling into multilayered arteries paired with peripheral nerves. This mature physiological competency was attained by targeting mesenchymal cells rather than endothelial cells, a finding that could inform strategies for therapeutic angiogenesis and tissue engineering.     

A constrained mixture model for developing mouse aorta

Mechanical stresses influence the structure and function of adult and developing blood vessels. When these stresses are perturbed, the vessel wall remodels to return the stresses to homeostatic levels. Constrained mixture models have been used to predict remodeling of adult vessels in response to step changes in blood pressure, axial length and blood flow, but have not yet been applied to developing vessels. Models of developing blood vessels are complicated by continuous and simultaneous changes in the mechanical forces. Understanding developmental growth and remodeling is important for treating human diseases and designing tissue-engineered blood vessels. This study presents a constrained mixture model for postnatal development of mouse aorta with multiple step increases in pressure, length and flow. The baseline model assumes that smooth muscle cells (SMCs) in the vessel wall immediately constrict or dilate the inner radius after a perturbation to maintain the shear stress and then remodel the wall thickness to maintain the circumferential stress. The elastin, collagen and SMCs have homeostatic stretch ratios and passive material constants that do not change with developmental age. The baseline model does not predict previously published experimental data. To approximate the experimental data, it must be assumed that the SMCs dilate a constant amount, regardless of the step change in mechanical forces. It must also be assumed that the homeostatic stretch ratios and passive material constants change with age. With these alterations, the model approximates experimental data on the mechanical properties and dimensions of aorta from 3- to 30-day-old mice.     

Functional comparison of the human isolated femoral artery, internal mammary artery, gastroepiploic artery, and saphenous vein

Human femoral, internal mammary, and gastroepiploic arteries and saphenous veins are used as bypass grafts for coronary surgery or for reconstruction in arterial occlusive disease. We have characterized the contractile responses of these vessels to various agents that are liberated during cardiac or vascular surgery. In organ baths, U46619 (a stable thromboxane A2 mimetic), norepinephrine, endothelin-1, angiotensin II, and KCl caused concentration-dependent contractions in all vessels tested. Leukotriene C4 did not induce any contraction in the arteries, whereas a contraction was obtained in the saphenous vein rings. U46619 induced the most powerful contraction in all vessels tested. The pD2 values for each agent did not differ among the different vessels. When responses were expressed as a percentage of KCl-induced contraction, the contraction of endothelin-1 (151+/-5%) and leukotriene C4 (43+/-5%) was more significant on saphenous veins than on arteries. In conclusion, thromboxane A2 appears to be the most potent endogenous constricting agent on different human vascular beds. Our second finding is that saphenous veins are more sensitive to contract to leukotriene C4 and endothelin-1 than arteries. These properties may influence early and (or) long-term vein graft patency.     

Recruitment of smooth muscle cells and arterial vasomotion

Investigating the recruitment and synchronization of smooth muscle cells (SMCs) is the key to understanding the physical mechanisms leading to contraction and spontaneous diameter oscillations of arteries, called vasomotion. We improved a method that allows the correlation of calcium oscillations (flashing) of individual SMCs with mean calcium variations and arterial contraction using confocal microscopy. Endothelium-stripped rat mesenteric arteries were cut open, loaded with dual calcium fluorescence probes, and stimulated by increasing concentrations of the vasoconstrictors phenylephrine (PE) and KCl. We found that the number and synchronization of flashing cells depends on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells flash asynchronously and no local contraction is detected. At medium concentration, recruitment of cells is complete and synchronous, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous calcium oscillations and fully contracted vessels, whereas high concentration of KCl leads to a sustained nonoscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling rat mesenteric artery contraction and vasomotion.     

Identification and functional response of interstitial Cajal-like cells from rat mesenteric artery

Cells with irregular shapes, numerous long thin filaments, and morphological similarities to the gastrointestinal interstitial cells of Cajal (ICCs) have been observed in the wall of some blood vessels. These ICC-like cells (ICC-LCs) do not correspond to the other cell types present in the arterial wall: smooth muscle cells (SMCs), endothelial cells, fibroblasts, inflammatory cells, or pericytes. However, no clear physiological role has as yet been determined for ICC-LCs in the vascular wall. The aim of this study has been to identify and characterize the functional response of ICC-LCs in rat mesenteric arteries. We have observed ICC-LCs and identified them morphologically and histologically in three different environments: isolated artery, freshly dispersed cells, and primary-cultured cells from the arterial wall. Like ICCs but unlike SMCs, ICC-LCs are positively stained by methylene blue. Cells morphologically resembling methylene-blue-positive cells are also positive for the ICC and ICC-LC markers α-smooth muscle actin and desmin. Furthermore, the higher expression of vimentin in ICC-LCs compared with SMCs allows a clear discrimination between these two cell types. At the functional level, the differences observed in the variations of cytosolic free calcium concentration of freshly dispersed SMCs and ICC-LCs in response to a panel of vasoactive molecules show that ICC-LCs, unlike SMCs, do not respond to exogenous ATP and [Arginine]⁸-vasopressin.