Stephen M. Schwartz

The Intima: Soil for Atherosclerosis and Restenosis

Thirty years ago, John French wrote a seminal review describing the unique properties of the arterial intima.1 His major point was that the smooth muscle cells of the intima have a unique morphology (Fig 1⇓). French also pointed out that intimal formation appeared during normal development and aging as well as in the response of arteries to almost any imaginable injury, including atherosclerosis. Figure 1. Top left, Histology of intima vs media. Coronary artery is from an adult patient with idiopathic dilated cardiomyopathy. In the absence of atherosclerosis, there remains diffuse intimal hyperplasia. The intima is several cell layers thick and composed of smooth muscle cells and is separated from the distinct media by the internal elastic lamina (arrows) (hematoxylin and eosin, original magnification ×200). Top right, Advanced atherosclerosis of the left circumflex coronary artery. This lesion was treated by percutaneous balloon angioplasty 23 days before the patient’s death. Note the complex nature of the lesion with a distinct fibrous cap (FC), intimal hyperplasia (IHP), intramural hemorrhage (H), and a necrotic core (NC) composed of inflammatory cells and accumulated lipid (hematoxylin and eosin, original magnification ×100). Bottom left, Myxomatous tissue. Directional coronary atherectomy specimen from a restenotic lesion shows stellate-shaped smooth muscle cells. Smooth muscle cells with this appearance are often regarded as evidence of proliferation. Note the homogeneity of these cells and the absence of inflammatory and endothelial cells in this region (hematoxylin and eosin, original magnification ×200). Bottom right, Directional coronary atherectomy specimen from a primary lesion. Again, note the presence of stellate-shaped smooth muscle cells but a denser connective tissue matrix (hematoxylin and eosin, original magnification ×200). This article attempts to update French’s review. We will discuss the developmental origins of the intima and suggest that the arterial intima is a distinct tissue with a long and …

Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall.

The present study was undertaken to explore the possibility that neointimal smooth muscle cells, the characteristic cells of restenosis and atherosclerosis, are selectively stimulated to replicate by a hypertensive stimulus. Angiotensin II (AII) was infused by osmotic minipumps for 2 weeks in 4.5-month-old rats. Group A received AII (200 ng/min) 2 weeks after a balloon catheter-induced injury of the thoracic aorta and left common carotid artery. Group B received only AII, group C only balloon denudation, and group D neither balloon injury nor AII. During the AII or Ringers solution infusion, all animals received [3H]thymidine via a second minipump to measure DNA synthesis. AII increased the systolic pressure by more than 40 mm Hg. AII significantly increased DNA synthesis in the media of the carotid artery from 0.2 +/- 0.2% in group C to 2.5 +/- 1.5% in group A (mean +/- SD, n = 5 or 6). DNA synthesis in the neointima of the carotid artery significantly increased with AII from 4.8 +/- 4.2% in group C to 19.8 +/- 13.9% in group A. Cross-sectional area of the neointima almost doubled during AII infusion, and it increased approximately 25% in the media. Comparable results were obtained in the aorta. In a second experiment, AII was infused (125 ng/min) for 2 weeks in 11-week-old rats. Concomitantly, [3H]thymidine was given. Control rats received Ringers solution and [3H]thymidine in their pumps. Blood pressures were elevated to the same extent as in the older animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal myoblast transplantation for repair of myocardial necrosis

Myocardial infarcts heal by scarring because myocardium cannot regenerate. To determine if skeletal myoblasts could establish new contractile tissue, hearts of adult inbred rats were injured by freeze-thaw, and 3-4.5 x 10(6) neonatal skeletal muscle cells were transplanted immediately thereafter. At 1 d the graft cells were proliferating and did not express myosin heavy chain (MHC). By 3 d, multinucleated myotubes were present which expressed both embryonic and fast fiber MHCs. At 2 wk, electron microscopy demonstrated possible satellite stem cells. By 7 wk the grafts began expressing beta-MHC, a hallmark of the slow fiber phenotype; coexpression of embryonic, fast, and beta-MHC continued through 3 mo. Transplanting myoblasts 1 wk after injury yielded comparable results, except that grafts expressed beta-MHC sooner (by 2 wk). Grafts never expressed cardiac-specific MHC-alpha. Wounds containing 2-wk-old myoblast grafts contracted when stimulated ex vivo, and high frequency stimulation induced tetanus. Furthermore, the grafts could perform a cardiac-like duty cycle, alternating tetanus and relaxation, for at least 6 min. Thus, skeletal myoblasts can establish new muscle tissue when grafted into injured hearts, and this muscle can contract when stimulated electrically. Because the grafts convert to fatigue-resistant, slow twitch fibers, this new muscle may be suited to a cardiac work load.

Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation.

Mesangial cell proliferation is common in glomerulonephritis but it is unclear if proliferation is associated with any in vivo alteration in phenotype. We investigated whether mesangial of mesangial proliferative nephritis induced with antibody to the Thy-1 antigen present on mesangial cells. At day 3 glomeruli displayed de novo immunostaining for alpha-smooth muscle actin in a mesangial pattern, correlating with the onset of proliferation, and persisting until day 14. An increase in desmin and vimentin in mesangial regions was also noted. Immunoelectron microscopy confirmed that the actin-positive cells were mesangial cells, and double immunolabeling demonstrated that the smooth muscle actin-positive cells were actively proliferating. Northern analysis of isolated glomerular RNA confirmed an increase in alpha and beta/gamma actin mRNA at days 3 and 5. Complement depletion or platelet depletion prevented or reduced proliferation, respectively; these maneuvers also prevented smooth muscle actin and actin gene expression. Studies of five other experimental models of nephritis confirmed that smooth muscle actin expression is a marker for mesangial cell injury. Thus, mesangial cell proliferation in glomerulonephritis in the rat is associated with a distinct phenotypic change in which mesangial cell assume smooth muscle cell characteristics.

Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques.

We studied death of human vascular smooth muscle cells derived from coronary plaques and normal coronary arteries and aorta. Cells from normal arteries underwent death only upon removal of serum growth factors. In contrast, plaque-derived cells died even in high serum conditions, and death increased after serum withdrawal. Death was characteristically by apoptosis in both normal and plaque-derived cells, as determined by time-lapse videomicroscopy, electron microscopy, and DNA fragmentation patterns. IGF-1 and PDGF were identified as potent survival factors in serum, whereas EGF and basic fibroblast growth factor had little effect. Stable expression of bcl-2, a protooncogene that regulates apoptosis in other cell lines, protected smooth muscle cells from apoptosis, although there was no detectable difference in endogenous bcl-2 expression between cells from plaques or normal vessels. We conclude that apoptosis of human vascular smooth muscle cells is regulated by both specific gene products and local cytokines acting as survival factors. Apoptosis may therefore regulate cell mass in the normal arterial wall and the higher rates of apoptosis seen in plaque smooth muscle cells may ultimately contribute to plaque rupture and breakdown and thus to the clinical sequelae of atherosclerosis.

Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for vascular smooth muscle cells (SMC) in vitro, but its activities in vivo remain largely undefined. We infused recombinant PDGF-BB (0.01-0.30 mg/kg per d i.v.) into rats subjected to carotid injury. PDGF-BB produced a small increase (two- to threefold) in medial SMC proliferation. More importantly, PDGF-BB greatly increased (20-fold) the intimal thickening and the migration of SMC from the media to the intima during the first 7 d after injury. These data provide support for the hypothesis that PDGF, and perhaps other platelet factors, might play an important role in the movement of mesenchymal cells into zones of injury undergoing repair.

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques.

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.

Renal injury from angiotensin II-mediated hypertension.

Angiotensin II (Ang II)-mediated hypertension induces vascular smooth muscle cell hypertrophy and hyperplasia in systemic blood vessels, but the effects of Ang II on the intrinsic cell populations within the kidney have been less well characterized. We infused Ang II for 14 days into rats by minipump at doses (200 ng/min) that resulted in moderate hypertension (mean systolic blood pressure 156-172 mm Hg). Small renal arterial vessels of Ang II-infused rats demonstrated focal injury with fibrinoid necrosis and medial hyperplasia, whereas the glomerular capillaries demonstrated only rare segmental hyalinosis. Proliferation of vascular smooth muscle cells was pronounced (fourfold to 20-fold increase in [3H]thymidine incorporation) as opposed to a minimal proliferation of glomerular cells in Ang II-infused rats. In contrast, the principal effect of Ang II in glomeruli was to increase the expression of alpha-smooth muscle actin by mesangial cells and desmin by visceral glomerular epithelial cells. Ang II-infused rats also developed focal tubulointerstitial injury, with tubular atrophy and dilation, cast formation, an interstitial monocytic infiltrate, and mild interstitial fibrosis with increased type IV collagen deposition. The injury was associated with a proliferation of distal tubule, collecting duct, and interstitial cells as determined by immunostaining for proliferating cell nuclear antigen, and was accompanied by an increase in platelet-derived growth factor B-chain messenger RNA in the area of interstitial injury as localized by in situ hybridization. Renal interstitial cells also underwent phenotypic modulation in which they expressed alpha-smooth muscle actin. Vehicle-infused control rats displayed no tubular injury, proliferation, or phenotypic modulation. Thus, Ang II in doses that cause moderate hypertension induces marked vascular, glomerular, and tubulointerstitial injury with cell proliferation, leukocyte recruitment, phenotypic modulation with the upregulation of proteins normally associated with smooth muscle cells, and interstitial fibrosis.

The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.

Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions.

Anal cancer incidence and survival: The Surveillance, Epidemiology, and End Results experience, 1973–2000

Anal cancer is a rare malignancy of the anogenital tract that historically has affected women at a greater rate than men.