Cellular proliferation in atherosclerosis and hypertension

We have tried to compare the proliferative responses seen in two vascular diseases: atherosclerosis and hypertension. Both diseases involve endothelial injury and proliferation, but our knowledge of this phenomenon is just beginning to emerge. In atherosclerosis the best evidence is that denudation does not occur in the normal young animal. Man, however, ages over a much longer time than our usual animal models, and the study of denudation during the chronic progression of atherosclerotic lesions remains to be done. We need to consider the possibility that repetitive, small lesions may occur at sites of endothelial turnover. We also need to know more about the possible role of nondenuding injuries, including death of endothelial cells in situ and the apparent increased stickiness of endothelial cells and monocytes during the early stages of hypercholesterolemia. The role of endothelial injury in hypertension also needs more study. We know that extensive denudation and thrombosis occur in small vessels subjected to high blood pressure. It is highly probable that release of PDGF occurs at these sites, possibly accounting for the characteristic hyperplasia seen in malignant hypertension. Whether this process is related to the more subtle changes in vessel wall mass seen in chronic hypertension remains unknown. Finally, there are remarkable differences in the proliferative behavior of the smooth muscle cells themselves in these two diseases. Hypertensive vascular disease is, in large part, a disease of the media. Atherosclerosis is characterized by intimal hyperplasia. Injury results in migration of smooth muscle cells from the media and cell division in the intima. It is possible to identify chemotactic factors using putative atherosclerosis risk factors or normal components of serum. This has already been done for one component of lesion formation, PDGF, and there is a report of a monocyte chemotactic factor released by smooth muscle cells. Factors released by other components of lesions may be of considerable interest. In contrast, changes in hypertension occur within a more orderly preservation of vessel wall structure. The wall thickens, but this occurs by increased synthesis of cell mass in the media. The cells themselves do not even divide, but they undergo a form of amitotic replication of their DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

The emerging cellular pathobiology of atherosclerosis.

The purpose of this paper is to present a brief coordinated overview of the recent results of research at the artery cell level which appear to have the greatest impact on the rapidly improving understanding of the pathogenesis of atherosclerosis in humans. The majority of these studies have employed in vitro methods and utilized the tools of modern cellular and molecular biology. These include microdissection; cell separation; tissue or cell culture; enzyme, lipid and protein chemistry as well as immunochemistry, ultrastructural visualization, cell organelle and membrane fractionation and the use of genetic markers. With these tools it is possible to study the interaction of the major space-occupying cells of the atherosclerotic plaque (especially the arterial smooth muscle cells) with many of the blood components, especially the lipoproteins and the other serum factors that appear to influence cell division. This direction of study appears to usher in a new era of atherosclerosis research.     

Cell proliferation, cell death and aging

An integrated view of the processes which most likely play a critical role in the aging process at the cellular level is proposed. Cells are continuously exposed to a variety of internal and external stressors, potentially dangerous for the maintenance of the functional integrity of the cell (UV and gamma radiation, heat, oxygen free radicals, glucose, bacteria, viruses). In the course of evolution a number of mechanisms [DNA repair, production of heat shock and other stress proteins, enzymatic and non-enzymatic antioxidant defence systems, poly(ADP-ribose) polymerase activation] have emerged which allow the cell to cope with such a variety of potentially harmful agents. These mechanisms are in fact interconnected and constitute a network of cellular defence systems. It is suggested that they play a physiological role, being involved in the control of gene expression. A failure of these mechanisms does not allow the cell to maintain homeostasis and has profound consequences as far as two of the major programs of the cell are concerned, i.e. cell proliferation and cell death. Recent data suggesting that these are two physiologically active phenomena tightly linked and regulated are examined. Thus, activation of cell cycle related genes and active inhibition of suicide genes appear to be a part of an integrated process. Conversely, deprivation of growth factors seems able to induce an active process of programmed cell death characterized by Ca++,Mg+(+)-dependent endonuclease activity and DNA fragmentation (apoptosis). Similar phenomena have been shown to accompany the terminal differentiation process in several cellular systems. The understanding of the factors which favour or prevent cell death (a phenomenon which has been recognized as one of the most important in fetal development and morphogenesis) will help to unravel and eventually to manipulate the aging process. In an evolutionary perspective, cell senescence appears to be the price paid to avoid unlimited capability of proliferation, i.e. cell transformation and cancer.     

Molecular and cellular imaging of atherosclerosis: emerging applications.

Molecular imaging studies have shed light on important biological aspects of atherosclerosis, and are now entering the clinical arena for the detection of clinical atheroma. This review first discusses fundamental principles regarding the rationale for and development of molecular imaging technologies for investigating atherosclerosis. Next, we highlight clinically promising imaging strategies that illuminate key biological aspects of atherosclerosis, including macrophage activity, protease activity, lipoprotein presence, apoptosis, and angiogenesis. We envision that several molecular imaging approaches will become important adjuncts to the clinical management of high-risk atherosclerosis.     

Necrotic cell death in atherosclerosis

Necrosis is a type of cell death characterized by a gain in cell volume, swelling of organelles, rupture of the plasma membrane and subsequent loss of intracellular contents. For a long time, the process has been considered as a merely accidental and uncontrolled form of cell death, but accumulating evidence suggests that it can also occur in a regulated fashion. Morphological studies using transmission electron microscopy indicate that the vast majority of dying cells in advanced human atherosclerotic plaques undergo necrosis. Various stimuli in the plaque including high levels of oxidative stress, depletion of cellular ATP, impaired clearance of apoptotic cells and increased intracellular calcium may cause necrotic death. Although the role of necrosis in atherosclerosis remains ill-defined, a growing body of evidence suggests that necrotic death stimulates atherogenesis through induction of inflammation and enlargement of the necrotic core. In addition, necrosis contributes to plaque instability by releasing tissue factor, matrix degrading proteases and pro-angiogenic compounds. Therapeutic agents against necrosis are limited, but efforts have recently been made to inhibit the necrotic pathway or its pro-inflammatory effects.     

Cell proliferation in human atherosclerosis

Despite the assumed proliferative nature of human atherosclerosis, very few studies exist that measure the actual proliferative rates in human arterial tissue. Recent proliferation-specific antibodies show very low rates of cell proliferation, as might be expected for a disease that takes numerous years to become clinically evident. These levels of proliferation are similar to animal models of hypercholesterolemia-induced atherosclerosis, and are unlike other models of acute mechanical injury to the artery wall. Proliferative activity in atherosclerotic plaques is seen among both smooth muscle cells and mononuclear inflammatory cells.     

Oestriol, oestradiol-17beta and the proliferation and death of uterine cells.

It has been suggested that oestriol protects against breast cancer, because in some experiments on uterine growth it is only weakly active, and partially inhibits the effects of oestradiol-17beta. When its effects are measured 24 h after a single injection, oestriol behaves as a typical impeded oestrogen with low potency and a flat dose-response line. This does not result from failure to stimulate certain critical stages of growth but from failure to sustain the products of growth. We found that oestriol induced all phases of uterine growth including DNA synthesis and cell division. It was as effective as oestradiol in stimulating early increases in protein synthesis and uterine weight, and half as effective in stimulating epithelial cells to replicate DNA and divide. However, epithelial cell numbers did not increase after a single injection of oestriol because cell death rate increased at the same time as mitotic rate, apparently as a result of the more rapid loss of oestriol from the uterus. Repeated injections of oestriol prevented premature cell death and produced as much uterine hypertrophy and hyperplasia as oestradiol-17beta. These results support the thesis that the oestrogenic potency of a substance is largely determined by the duration of its occupation of receptors. Thus in situations of continuous production, (e.g. pregnancy) oestriol would be as active as oestradiol and unlikely to exert any significant 'buffering' or protective action. The findings are also discussed in relation to a new model for the regulation of cell proliferation.     

Apoptotic cell death and efferocytosis in atherosclerosis

Apoptotic cell death is an important feature of atherosclerotic plaques, and it seems to exert both beneficial and detrimental effects depending on the cell type and plaque stage. Because late apoptotic cells can launch proatherogenic inflammatory responses, adequate engulfment of apoptotic cells (efferocytosis) by macrophages is important to withstand atherosclerosis progression. Several efferocytosis systems, composed of different phagocytic receptors, apoptotic ligands, and bridging molecules, can be distinguished. Because phagocytes in atherosclerotic plaques are very much solicited, a fully operative efferocytosis system seems to be an absolute requisite. Indeed, recent studies demonstrate that deletion of just 1 of the efferocytosis pathways aggravates atherosclerosis. This review discusses the role of apoptosis in atherosclerosis and general mechanisms of efferocytosis, to end with indirect and direct indications of the significance of effective efferocytosis in atherosclerosis.     

Cellular and molecular mechanisms of atherosclerosis with mouse models

Recently, there has been an explosion in the number of in vivo studies using genetically engineered mouse models. Atherosclerosis research using mice began with the invention of traditional atherosclerotic mice including low-density lipoprotein receptor knockout (LDLR(-/-)) and apolipoprotein E knockout (apoE(-/-)) mice, which provided tremendous progress in atherosclerosis research. Since then, a number of modified atherosclerotic mouse models have been reported to generate lesions that more closely characterize human atherosclerotic lesions. Those modifications include inflammation, hypertension, proteinases and extracellular matrix, glucose metabolism, and immune systems. This article focuses on various kinds of mouse models with atherosclerosis and their contributions to the current advances of research.     

Cellular immunity in congestive cardiomyopathy. The normal cellular immune response.

In vivo and in vitro tests of cellular immunity were studied in patients with congestive cardiomyopathy to determine whether these patients have normal or depressed cell mediated immunity to common environmental antigens and mitogens. No abnormality was found, but this does not exclude the possibility that transient depression of cell mediated mechanisms occurs early in the illness before clinical presentation.     

Cellular proliferation induced in the lung by cadmium aerosol.

Rats were exposed to a polydispersed aerosol of 0.1 per cent cadmium chloride in physiologic saline for a single period of 2 hours, and the evolution of damage was followed for a 10-day period. Control animals were unexposed rats and rats exposed to an aerosol of physiologic saline. Wet lung weight doubled by the fourth day in animals exposed to cadmium chloride, but not in the control groups. Apart from a transient increase in fluid content at 1 day, there was no evidence that the lung weight gain was due to inflammatory edema. Uptake of [3H]thymidine markedly increased during the first four days after exposure to cadmium chloride. Light autoradiography showed that [3H]thymidine labeling occurred almost exclusively in Type II alveolar cells at 1 day: later, the label appeared in interstitial cells and cells lying free within alveoli. These changes correlated well (r=0.70) with the increase in deoxyribonucleic acid on the fourth day after exposure. Uptake of [3H]thymidine and deoxyribonucleic acid content in rats exposed to cadmium chloride showed a significant difference from the control groups (P less than 0.001). These experiments demonstrate that exposure to cadmium chloride aerosol evokes a wave of cellular proliferation in rat lung. This finding is of interest, because heavy industrial exposure to cadmium in humans is known to be associated with severe physiologic impairment and anatomic damage.     

Aging and atherosclerosis: mechanisms, functional consequences, and potential therapeutics for cellular senescence

Atherosclerosis is classed as a disease of aging, such that increasing age is an independent risk factor for the development of atherosclerosis. Atherosclerosis is also associated with premature biological aging, as atherosclerotic plaques show evidence of cellular senescence characterized by reduced cell proliferation, irreversible growth arrest and apoptosis, elevated DNA damage, epigenetic modifications, and telomere shortening and dysfunction. Not only is cellular senescence associated with atherosclerosis, there is growing evidence that cellular senescence promotes atherosclerosis. This review examines the pathology of normal vascular aging, the evidence for cellular senescence in atherosclerosis, the mechanisms underlying cellular senescence including reactive oxygen species, replication exhaustion and DNA damage, the functional consequences of vascular cell senescence, and the possibility that preventing accelerated cellular senescence is a therapeutic target in atherosclerosis.     

Cytokine signaling for proliferation, survival, and death in hematopoietic cells

The survival, proliferation, and differentiation of hematopoietic cells are regulated by cytokines. In the absence of cytokines, hematopoietic cells not only stop proliferation, but undergo apoptosis. This strict dependency of hematopoietic cells on cytokines is an important mechanism that maintains the homeostasis of blood cells. Cytokines induce various intracellular signaling pathways by activating the receptor-associated Janus kinases (Jaks), and distinct signals are responsible for cell cycle progression and cell survival. Induction of signals for cell cycle progression without suppressing apoptosis results in apoptotic cell death, indicating the essential role of anti-apoptotic signaling for cell growth. In hematopoietic cells, Ras, a cellular protooncogen product, and phosphatidylinositol 3 kinase are involved in the suppression of apoptosis. Cytokine depletion not only turns off anti-apoptotic signaling, but also actively induces cell death by activating caspases, a distinct family of cysteine proteases. Alterations in the mechanisms of cytokine signaling for cell cycle progression and anti-apoptotic function are implicated in hematological disorders.     

Cellular immunity in congestive cardiomyopathy. The normal cellular immune response

In vivo and in vitro tests of cellular immunity were studied in patients with congestive cardiomyopathy to determine whether these patients have normal or depressed cell mediated immunity to common environmental antigens and mitogens. No abnormality was found, but this does not exclude the possibility that transient depression of cell mediated mechanisms occurs early in the illness before clinical presentation.     

Pathogen burden, inflammation, proliferation and apoptosis in human in-stent restenosis. Tissue characteristics compared to primary atherosclerosis

Pathogenic events leading to in-stent restenosis (ISR) are still incompletely understood. Among others, inflammation, immune reactions, deregulated cell death and growth have been suggested. Therefore, atherectomy probes from 21 patients with symptomatic ISR were analyzed by immunohistochemistry for pathogen burden and compared to primary target lesions from 20 stable angina patients. While cytomegalovirus, herpes simplex virus, Epstein-Barr virus and Helicobacter pylori were not found in ISR, acute and/or persistent chlamydial infection were present in 6/21 of these lesions (29%). Expression of human heat shock protein 60 was found in 8/21 of probes (38%). Indicated by distinct signals of CD68, CD40 and CRP, inflammation was present in 5/21 (24%), 3/21 (14%) and 2/21 (10%) of ISR cases. Cell density of ISR was significantly higher than that of primary lesions (977 +/- 315 vs. 431 +/- 148 cells/mm(2); p < 0.001). There was no replicating cell as shown by Ki67 or PCNA. TUNEL(+) cells indicating apoptosis were seen in 6/21 of ISR specimens (29%). Quantitative analysis revealed lower expression levels for each intimal determinant in ISR compared to primary atheroma (all p < 0.05). In summary, human ISR at the time of clinical presentation is characterized by low frequency of pathogen burden and inflammation, but pronounced hypercellularity, low apoptosis and absence of proliferation.     

Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes

During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell-cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, α-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.     

Cellular proliferation at sutured and sutureless colonic anastomoses

Elevated cellular proliferation in the vicinity of an anastomosis may explain the enhanced susceptibility to carcinogens. The aim of this study was to determine whether anastomotic cellular proliferation was altered by different suture materials and whether a rise in cell turnover also occurred after a sutureless closure. A transverse descending colon enterotomy was repaired with interrupted sutures of 5/0 silk (n=20), stainless steel (n=20), or Vicryl ^® (Ethicon, Inc., Somerville, NJ) (n=20) or by a sutureless technique (n=20). Using a stathmokinetic technique, crypt cell production rates (CCPR) were calculated at the anastomosis and in the adjacent colon at varying intervals between one week and six months after treatment. Overall colonic cellular proliferation appeared to be elevated at a sutured colotomy for at least three months (P<0.05). In contrast, no significant elevation in cellular proliferation was observed at sutureless anastomoses. The duration of elevated proliferative response varied among the sutures.