Julie H. Campbell

Replication of smooth muscle cells in vascular disease.

Smooth muscle proliferation has been recognized as central to the pathology of both major forms of vascular disease: atherosclerosis and hypertension. Recent advances in our knowledge of mechanisms of control of proliferation suggest that events occurring in adult animals may recapitulate portions of the developmental biology of the smooth muscle cell. This review attempts to consider the current state of knowledge of the mechanisms controlling smooth muscle proliferation in these two diseases, to put that knowledge into the context of what is known about smooth muscle biology, and to offer two hypotheses on the possible roles of smooth muscle developmental biology in manifestations of atherosclerosis and hypertension in adult humans.

Circulating Bone Marrow Cells Can Contribute to Neointimal Formation

To examine the source of smooth muscle-like cells during vascular healing, C57BL/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 106 nucleated bone marrow cells from congenic (Ly 5.1) male donors. Successful repopulation (88.4 ± 4.9%) by donor marrow was demonstrated in the female mice by flow cytometry with FITC-conjugated A20.1/Ly 5.1 monoclonal antibody after 4 weeks. The arteries of the female mice were then subjected to two types of insult: (1) The iliac artery was scratch-injured by 5 passes of a probe causing severe medial damage. After 4 weeks, the arterial lumen was obliterated by a cell-rich neointima, with cells containing α smooth muscle actin present around the residual lumen. Approximately half of these cells were of male donor origin, as evidenced by in situ hybridization with a Y-chromosome-specific probe. (2) In an organized arterial thrombus formed by inserting an 8-0 silk suture into the left common carotid artery, donor cells staining with α smooth muscle actin were found in those arteries sustaining serious damage but not in arteries with minimal damage. Our results suggest that bone marrow-derived cells are recruited in vascular healing as a complementary source of smooth muscle-like cells when the media is severely damaged and few resident smooth muscle cells are available to effect repair.

Cytodifferentiation and expression of alpha-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells. Correlation with cell density and proliferative state.

To investigate a possible correlation between cytodifferentiation, proliferation, and actin expression, smooth muscle cells from the 9-week-old rabbit aortic media were enzyme-dispersed into single cells and were plated in primary culture at different initial seeding densities. The volume fraction of myofilaments (Vv myo) in cells seeded moderately densely fell from 39.5% +/- 1.2% in the intact aortic media to 11.5% +/- 1.6% on Day 5, one day before the onset of logarithmic growth. The Vv myo remained low over the next 3 days, then began to rise as the density of cells increased, returning almost to the original levels after confluency and 1.84 cumulative population doublings (CPD). The expression of alpha-smooth muscle actin mRNA followed a similar time course of change, falling from 84.7% +/- 1.2% of total actin mRNA in freshly isolated cells to 54.0% +/- 6.5% on Day 5, returning to 87.5% +/- 0.5% after confluency. In these cultures, the alpha-smooth muscle actin protein content was 93.7% +/- 2.9% of total actin in freshly isolated cells, 68.7% +/- 3.1% on Day 5, and 73.3% +/- 2.5% 3 days after confluency. In densely seeded cultures, the Vv myo and expression of alpha-smooth muscle actin mRNA fell only slightly on Day 5 and rose to original levels upon confluency after 0.33 CPD. However, at the protein level, alpha-smooth muscle actin decreased on Day 5 and remained low on Day 12. The Vv myo, alpha-smooth muscle actin mRNA, and actin protein of sparsely seeded cells fell on Day 5 and then remained low throughout the culture period, including 5 days after confluency (Day 24), when the cells had undergone 5.37 CPD. Cells that were maintained subconfluent but quiescent on Day 7 in culture had the same low Vv myo, low alpha-actin mRNA expression, and low alpha-actin protein content as actively proliferating cells. The results show that Vv myo and alpha-smooth muscle actin mRNA undergo parallel changes during primary culture according to seeding density, but not to replication, and that alpha-smooth muscle actin protein decreases in culture then remains low irrespective of culture conditions.

Tissue-engineered blood vessels: alternative to autologous grafts?

Although vascular bypass grafting remains the mainstay for revascularization for ischemic heart disease and peripheral vascular disease, many patients do not have healthy vessels suitable for harvest. Thus, prosthetic grafts made of synthetic polymers were developed, but their use is limited to high-flow/low-resistance conditions because of poor elasticity, low compliance, and thrombogenicity of their synthetic surfaces. To fill this need, several laboratories have produced in vivo or in vitro tissue-engineered blood vessels using molds or prosthetic or biodegradable scaffolds, but each artificial graft has significant problems. Recently, conduits have been grown in the peritoneal cavity of the same animals in which they will be grafted, ensuring no rejection, in the short time of 2 to 3 weeks. Remodeling occurs after grafting such that the tissue is almost indistinguishable from native vessels. This conduit is derived from cells of bone marrow origin, opening new possibilities in vascular modeling and remodeling.

The role of smooth muscle cells in atherosclerosis.

The smooth muscle cells within the human atherosclerotic plaque differ from the cells of the media. These phenotype changes, the mechanisms responsible, and their possible relevance to the formation of the plaque are discussed.

Haemopoietic origin of myofibroblasts formed in the peritoneal cavity in response to a foreign body

This study utilized both in vivo and in vitro techniques to investigate whether cells of bone marrow origin can differentiate into smooth muscle-like cells (myofibroblasts) with contractile filaments and proteins. Female C57BL/6 mice expressing the Ly5.2 antigen on the surface of their haemopoietic cells had four pieces of silastic tubing (3 × 0.5 mm outer diameter) or boiled blood clot (2–3 mm diameter) placed in their peritoneal cavity. After 3, 5, 7 and 14 days (n = 4/group) the implants were removed and those that had remained free-floating were processed for light microscopy, immunohistochemistry and electron microscopy. In the first 3–5 days, rounded cells adhered to the entire surface of the tubing then flattened. These cells stained with fluoresceinated antibodies to Ly5.2 showing that they were derived from haemopoietic cells. By 14 days the cells had become elongated and multilayered in a collagen matrix, forming a thick tissue capsule around the tubing or boiled clot. They contained contractile filaments and stained with antibodies to α-smooth muscle actin but no longer stained for Ly5.2. A separate set of female C57BL/6 Ly5.2 mice were X-irradiated to destroy bone marrow then immediately transfused with 106 nucleated bone marrow cells taken from the femur and tibia of a congenic strain of male mice expressing the Ly5.1 allele. Eight of the female mice with successful engraftment (80–99%) had silastic tubing implanted in the peritoneal cavity. After 14 days, in situ hybridization with Y chromosome probe confirmed the male donor, and thus bone marrow, origin of the elongated cells that formed the capsule. In vitro studies showed that cells of the murine macrophage cell lines RAW 264.7 and J774 express α-smooth muscle actin after exposure to the cytokine γ-interferon in vitro. These data show that bone marrow-derived cells can differentiate into smooth muscle-like cells and raises the possibility that blood-derived cells may contribute to the development of fibro-proliferative vascular diseases such as atherosclerosis.

Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair

Macrophages participate in the restenosis process through the release of cytokines, metalloproteinases and growth factors. Studies of peritoneal granulation tissue suggest that macrophages may be precursors of myofibroblasts. This study examined the contribution of monocyte/macrophage lineage cells to neointimal cellular mass in a porcine model of thermal vascular injury. Thermal coronary artery injury caused medial smooth muscle cell necrosis and transformation of the media into an extracellular matrix barrier. The neointimal hyperplasia that developed over the injury sites was evaluated by light microscopy, electron microscopy and immunohistochemistry. At day 3, blood monocytes were adhered to the vessel wall and infiltrated the fibrotic media. At day 14, 42+/-3.9% of neointimal cells had a monocytic nuclear morphology and expressed macrophage-specific antigen SWC3 (identified by monoclonal antibody DH59B). Moreover, 9.2+/-1.8% of neointimal cells co-expressed SWC3 and alpha-smooth muscle actin and had ultrastructural characteristics intermediate between macrophages and myofibroblasts. At day 28, 10.5+/-3.5% of cells expressed SWC3 and 5.2+/-1.8% of cells co-expressed SWC3 and alpha-smooth muscle actin. This study indicates that hematopoietic cells of monocyte/macrophage lineage abundantly populate the neointima in the process of lesion formation and may be precursors of neointimal myofibroblasts after thermal vascular injury.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

PURPOSE The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.

Smooth muscle phenotypic expression in human carotid arteries. II. Atherosclerosis-free diffuse intimal thickenings compared with the media.

The volume fraction of synthetic organelles in smooth muscle cells from areas of atherosclerosis-free, diffuse intimal thickenings (DIT) and media of human carotid artery has been determined morphometrically. Tissue was obtained at autopsy from men and women aged 35 to 80 years, at 6 to 12 hours after death. With both sexes, the volume fraction of synthetic organelles in smooth muscle cells from the DIT was not significantly different from the volume fraction of synthetic organelles in cells of the subjacent media (0.25 and 0.21, respectively). This is in marked contrast to our earlier study in which the volume fraction of synthetic organelles in smooth muscle cells of DIT from a similar region of artery, but adjacent to atheromatous plaques, was highly significantly different from cells of the subjacent media (0.48 and 0.23, respectively). Only in regions near atherosclerotic plaques do the majority of smooth muscle cells of DIT express a different phenotype from cells of the media.

Rhythmic variations in pain, stiffness, and manual dexterity in hand osteoarthritis

Objective: To explore circadian variation in pain, stiffness, and manual dexterity in patients with hand osteoarthritis (OA). Methods: Twenty one patients with hand OA, as defined by ACR criteria (17 women, four men, mean age 62.2 years, range 52–74 years) self rated pain and stiffness on separate 10 cm horizontal visual analogue scales and performed bead intubation coordinometry (BIC) six times each day (on waking up, at bedtime, and every four hours in between) for 10 consecutive days. Each series (using data with the trend removed if there was a significant trend) was analysed for circadian rhythmicity by a cosine vector technique (single cosinor). With individual data expressed as the percentage of the mean, group rhythm characteristics at period 24 hours were summarised for each variable by population mean cosinor analysis. Results: Individual analyses identified significant circadian rhythms at p≤0.05 for pain (n=15/21), stiffness (n=16/20), and dexterity (n=18/21), and a significant circadian rhythm on a group basis was identified for pain (p=0.013), stiffness (p<0.001), and dexterity (p<0.001). Pain was least at 1610 and stiffness at 1618. Peak dexterity occurred in mid-afternoon at 1548 and occurred within the 95% confidence interval of least pain (1312–1800) and stiffness (1520–1732). Conclusions: Dexterity was influenced by the patients level of pain or stiffness, which changed systematically throughout the day. Similar results have been previously reported in 14 patients with rheumatoid arthritis where peak dexterity occurred at 1544 and at 1528 in 14 age and sex matched healthy controls. The predictability of rhythmic variation in pain, stiffness, and dexterity has implications for scheduling activities of daily living and for timing antirheumatic drug treatment.