DISEASE FOCUS: Atherosclerosis and vascular calcification

Overview of atherosclerosis

Cardiovascular disease accounts for approximately 56% of the total mortality in the over 65 age group and represents the single largest age-related cause of death (Brock et al, 1990, Mills et al). Atherosclerosis constitutes the single most important contributor to this increasing problem of cardiovascular disease. Atherogenesis is a complicated process which includes endothelial cell (EC) dysfunction, smooth muscle cell (SMC) proliferation and migration, recruitment of inflammatory cells, lipid and matrix accumulation and thrombus formation (Tuomisto et al 2005).

To better understand the pathological processes that occur with atherosclerosis, an understanding of the structure of arteries is required. Human arteries are composed of three layers, the intima, the media and the adventitia. The intima is the innermost layer of the artery, composed of EC’s, SMC’s, macrophages and extracellular matrix (ECM) components. An internal elastic lamina separates the intima from the media, which is made up mainly of SMC. The adventitia is separated from the media by external elastic lamina and is mainly composed of fibroblasts and connective tissue.

The initiation and progression of atherosclerotic plaques generally takes place over many years during which the affected individual remains symptom free. Therefore, when a patient becomes symptomatic, the disease is already well established. These plaques occur at specific sites within arteries and these sites are dictated by fluid shear stress, the frictional force generated by blood flow over the vascular endothelium (Hwang et al, 2003). Regions of branched and curved arteries experience the greatest disturbed blood flow and it is at these sites that high incidences of plaque formation is found (VanderLaan et al, 2004). Relatively straight arteries however, experience the least shear stress and are usually protected from plaque development. Explanations for why high fluid stress sites are more “lesion-prone” is currently speculative.

The initial factors which result in the initiation of plaque formation are currently unknown. A common hypothesis is that plaque formation occurs as a result of EC damage leading to cellular dysfunction (Shimokawa, 1999, Davignon and Ganz, 2004). The source of the initial damage to EC’s is also currently unknown, but hypertension, viruses, toxins, smoking have all been suggested. Cellular dysfunction results in subsequent recruitment and accumulation of leukocytes and monocytes which would otherwise have resisted any adhesive interactions (Bobryshev et al, 2005). These adhered monocytes then differentiate into macrophages, engulf lipids, become foam cells and form fatty streaks. As the progression of the plaque continues, SMC’s migrate from the intima and synthesis extracellular matrix proteins in the intima (Boyle et al, 1997). Progressive macrophage accumulation, SMC migration and proliferation and extracellular matrix protein synthesis result in the formation of an advanced lesion.

A schematic representation of the structure of an artery, showing the intima, media and adventitia (commons.wikimedia.org)

Cellular senescence in disease states

In some instances, cellular senescence is thought to contribute to the development and/or progression of age-related disease, but in others, the presence of disease may accelerate the accumulation of senescent cells.

A recent study has provided strong evidence to suggest that intervertebral disc degeneration, a major cause of low back pain, is due to accelerated cellular senescence (Le Maitre et al, 2007). Cells isolated from normal and degenerate human tissue were assessed for mean telomere length, SA-β-Gal, and replicative potential. Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. SA-β-Gal staining was not observed in non-degenerative patients unlike cells from degenerative discs which did exhibit 10-12% SA-β-Gal staining and decrease in replicative potential. However, the factors which may have led to accelerated senescence in this instance was not discussed. There are three possible reasons why cellular senescence was accelerated in this instance; (1) Unknown factors resulted in the damage and removal of cells, resulting in cell turnover for replacement, (2) ROS were involved causing stress induced premature senescence (SIPS) or (3) telomeres in these cells for some unknown reason started off shorter than normal, meaning less cell turnover is required for the appearance of senescent cells.

Other studies have shown a correlation between disease states and the presence of senescent cells in vivo. SA-β-Gal staining was used to detect senescent cells in normal liver, liver with chronic hepatitis C and hepatocellular carcinoma (HCC) (Paradis et al, 2001). They found senescent cells present in 3 of 15 (20%) normal livers tested, 16 of 32 (50%) in livers with chronic hepatitis C and in 6 of 10 (60%) livers with HCC. The presence of senescent cells in normal livers was found to be associated with old age. Interestingly, the presence of senescent cells in non-tumoural tissues was strongly correlated with the presence of HCC in the surrounding liver. This demonstrates not only that the ageing of one tissue can have a direct impact on another but also as suggested by Judith Campisi, senescent cells may contribute to carcinogenesis (Campisi, 1997).

Another study looked at cellular senescence in human benign prostatic hyperplasia (BPH) specimens (Choi et al, 2000). BPH is a disease associated with an abnormal growth of the adult prostate that begins mid to late life. Results from this study found that 40% of the analysed samples showed positive staining for SA-β-Gal and only in the epithelial cells. A high prostate weight (> 55g) was found to correlate strongly with the expression of SA-β-Gal. Prostates weighing less than 55g tended to lack senescent epithelial cells. It was suggested that the accumulation of senescent epithelial cells may play a role in the development of prostatic enlargement associated with BPH. However, the accumulation of senescent cells in this case is likely to be a consequence of the disease, which may lead further to its progression. The enlargement of the prostate may be the result of unregulated stimulated proliferation, increasing cell turnover and consequently the appearance of senescent cells. This may explain why a stronger expression of SA-β-Gal is detected in prostates weighing more than 55g since they may have undergone more cellular divisions.

During the pathogenesis of type 2 diabetes, insulin resistance causes compensatory proliferation of pancreatic beta cells. This compensatory proliferation might accelerate cellular senescence contributing further to the progression of diabetes. To investigate this, one group used nutrient-induced diabetic mice to analyse beta cells for SA-β-Gal and the proliferation marker Ki67 (Sone and Kagawa, 2005). At 4 months, the proliferation of beta cells was 2.2 fold higher than in the control group. At 12 months, the frequency of Ki67 decreased to one-third that of the control and SA-β-Gal positive cells increased to 4.7 fold that of the control group. This increase in the senescent beta cell fraction correlated with insufficient insulin release, suggesting cellular senescence may contribute to diet-induced diabetes. In this instance it is difficult to determine whether senescence is the cause or the consequence of insulin resistance. It later appears to be a contributor but whether it is also the initiating factor is unknown.

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An increase in the number of senescent primary lung fibroblasts has also been shown to increase in patients with emphysema compared with normal controls (Muller et al, 2006). An average of 4% of cells from control patients stained positive for SA-β-Gal compared to an average of 16% in patients with emphysema. It is possible that long-term exposure to tobacco smoke accelerates the formation of senescent cells, which subsequently may lead to loss of elasticity of the lung tissue, destruction of structures supporting the alveoli, and destruction of capillaries feeding the alveoli observed with emphysema. One study has shown for example that cigarette smoke induces senescence in alveolar epithelial cells (Tsuji et al, 2004).

Another study used senescent associated p16 instead of SA-β-Gal to detect senescent cells in kidneys with glomerular disease (GD) (Sis et al, 2007). Glomerular diseases include many conditions which fall into two major categories; Glomerulonephritis describes the inflammation of the membrane tissue in the kidney that serves as a filter, separating wastes and extra fluid from the blood. Glomerulosclerosis is the scarring or hardening of the tiny blood vessels within the kidney. This study found an increased expression of the nuclear p16 in samples with GD compared with normal. Independently, older age and interstitial inflammation was associated with increased expression of nuclear p16. Since senescent cells adopt a pro-inflammatory phenotype, their presence may be a contributing factor in inflammation observed in glomerulonephritis.

All these examples demonstrate the presence of senescent cells not only in vivo, but more specifically in disease states. This suggests that the accumulation of senescent cells in normal tissues is the result of injuries to those tissues or in some cases unregulated stimulation of proliferation. This suggests that if no injuries occurred as a consequence of disease, environmental factors or by normal biological/mechanical wear and tear, ageing of mitotic tissues would be greatly reduced.

Whether cellular senescence is the cause or the consequence of some diseases is yet to be answered.

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www.springerlink.com/index/JW847G541L435103.pdf

Physiological and pathological consequences of cellular senescence

Replicative capacity of cells from disease states

The gradual appearance of senescent cells may contribute to the development of age-related disease. However, the presence of disease by other mechanisms may result in accelerated senescence. Disease may cause tissue damage which leads to cellular turnover for the purpose of replacing lost cells. This exhausts the replicative capacity of the cells and accelerates the appearance of senescent cells. For example Goldstein and co-workers (1978) looked at the replicative lifespan of fibroblasts from normal, prediabetic, diabetic donors. Diabetes mellitus is a common genetically determined disorder associated with reduced life expectancy. This study confirmed earlier findings that there is an inverse correlation between donor age and replicative lifespan, but emphasised the importance of physiological state of the donors. Normal cell strains showed significantly better growth capacity than diabetic and prediabetic cells. The results indicated that with an increasing predisposition to diabetes, there is a progressive decrease in replicative capacity.

Another group investigating atherosclerosis took vascular smooth muscle cells (VSMC) from human atherosclerotic plaques and grew them in culture (Bennett et al, 1998). Results showed that VSMCs taken from plaques have lower rates of proliferation and underwent senescence earlier than cells derived from normal vessels.

A more recent study looked at the replicative capacity of osteoblasts in Rheumatoid arthritis (RA) compared with Osteoarthritis (OA) (Yudoh et al, 2000). The results indicated that the replicative capacity of osteoblasts decreased gradually with donor age and this decrease was higher in RA patients than with OA patients at any donor age. They also reported an increase in senescent osteoblastic cells with age in both groups in which the rate of expression of senescent cells was higher in RA patients than with age-matched OA patients.

Tesco et al (1993) looked at the replicative capacity of fibroblasts in patients with familial Alzheimer’s disease (FAD) to examine whether features compatible with a systemic premature aging were present. Data showed that there was no significant difference in replicative capacity of fibroblasts between FAD patients and controls. This is not a surprising result, since the fibroblasts studied are unrelated to the development of FAD and if features of premature ageing were present they would have most likely manifested themselves as other diseases other than just Alzheimer’s. For example, Werner’s syndrome is a premature ageing disorder which displays a multitude of age-related afflictions including diabetes and heart disease (Kipling and Faragher, 1997). When fibroblasts were taken from patients with Werner’s syndrome and grown in culture, the number of population doublings achieved was smaller compared with normal cells of a similar chronological age (Martin et al, 1970)

These studies suggest that disease is an important factor contributing to the exhaustion of the replicative capacity of cells. However, it is possible that some diseases arise as a result of the gradual increase in senescent cells with time. It is also possible that unknown factors result in accelerated senescence, which subsequently manifests itself as a biological impairment or disease.

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Factors, other than disease, which may contribute to cellular injury and cell loss, may be environmental such as UV radiation, chemical damage from smoking and foods, and normal biological damage from general wear and tear.