Medial Smooth Muscle Cell Number Research Articles

Tc-99m-labeled endothelin derivative for imaging of experimentally induced atherosclerosis

Objective: to characterize the potential of an endothelin derivative labeled with technetium-99m (Tc-99m) for the imaging of experimentally induced atherosclerosis. Methods: neointima of different cellularity and severity of stenosis was induced in 32 rabbits by balloon denudation followed by distinct dietary regimens and drug application. Angiograms and scintigrams after injection of the Tc-99m-labeled endothelin derivative were obtained. The aorta was dissected for autoradiography, sudan-III-staining, morphometry, and immunohistology. Results: the lesions induced could be detected in vivo (whole body scintigram) in all the animals 15 min after the injection of the Tc-99m endothelin derivative. Autoradiography revealed a strong relationship between tracer accumulation and sudan-III-staining of lesions. Accumulation of the endothelin derivative correlated with the number of neointimal smooth muscle cells (SMC), but not with the number of medial SMC, neointimal macrophages, and neointimal area. Conclusions: the results indicate that in vivo imaging of atherosclerosis with an endothelin derivative is a feasible method of detecting and characterizing atherosclerotic arterial wall lesions at early stages.

Smooth muscle cell matrix metalloproteinase production is stimulated via alpha(v)beta(3) integrin.

This study tests the hypothesis that alpha(v)beta(3) integrin receptors play a critical role in smooth muscle cell (SMC) migration after arterial injury and facilitate migration through the upregulation of matrix metalloproteinase (MMP) activity. We showed that beta(3) integrin mRNA was upregulated by SMCs in the balloon-injured rat carotid artery in coincidence with MMP-1 expression and early SMC migration. Treatment with the beta(3) integrin-blocking antibody F11 significantly decreased SMC migration into the intima at 4 days after injury, from 110.8+/-30.8 cells/mm(2) in control rats to 10.29+/-7.03 cells/mm(2) in F11-treated rats (P=0.008). By contrast, there was no effect on medial SMC proliferation or on medial SMC number in the carotid artery at 4 days. In vitro, we found that human newborn SMCs produced MMP-1 but that adult SMCs did not. This was possibly due to the fact that newborn SMCs expressed alpha(v)beta(3) integrin receptors, whereas adult SMCs did not. Stimulation of newborn (alpha(v)beta(3)+) SMCs with osteopontin, a matrix ligand for alpha(v)beta(3), increased MMP-1 production from 114.4+/-35.8 ng/mL at 0 nmol/L osteopontin to 232.5+/-57.5 ng/mL at 100 nmol/L osteopontin. Finally, we showed that stimulation of newborn SMCs with platelet-derived growth factor-BB and osteopontin together increased the SMC production of MMP-9. Thus, our results support the hypothesis that SMC alpha(v)beta(3) integrin receptors play an important role in regulating migration by stimulating SMC MMP production.

Vascular smooth muscle cell cytotoxicity and sustained inhibition of neointimal formation by fibroblast growth factor 2-saporin fusion protein.

Basic fibroblast growth factor (FGF2) is an important mediator of smooth muscle cell (SMC) proliferation following arterial injury that results in neointimal growth. The present study was designed to explore the effects of recombinant FGF2 linked to the ribosome-inactivating protein saporin-6 (rFGF2-SAP) on vascular SMC cytotoxicity and neointimal formation following arterial injury. Cultured rat aortic SMCs were exposed to various concentrations of rFGF2-SAP, FGF2, and saporin-6 (SAP). Incubation with rFGF2-SAP resulted in a decreased number of SMCs beginning at a concentration of 10(-9) mol/L. Significant cytotoxicity was observed with as little as a 30-minute exposure of SMCs to rFGF2-SAP. To evaluate the ability of rFGF2-SAP in an in vivo model to reduce neointimal formation, Sprague-Dawley rats underwent carotid artery balloon denudation and received an intravenous bolus of vehicle or 5, 10, 15, or 20 micrograms/kg rFGF2-SAP on 0, 3, 6, and 9 days after injury. Rats were euthanized at 14 days, and carotid arteries were analyzed by computerized morphometry. The threshold dose for a significant reduction in neointimal area by rFGF2-SAP was 15 micrograms/kg (47% reduction in neointima). When dosing was extended to include days 16, 19, and 22, the neointima was reduced 33% at 28 days (P = .048). rFGF2-SAP reduced neointima without associated medial thinning or arterial wall dilatation. To determine if rFGF2-SAP directly targets SMCs in vivo, rats underwent carotid injury and received either 15 micrograms/kg rFGF2-SAP or vehicle on day 0 and at 72 hours, with euthanasia at 78 hours after balloon denudation. Medial SMC number was reduced 46% in the rFGF2-SAP group. Tissue sections from arteries 3 days after balloon injury demonstrated rFGF2-SAP binding to medial SMCs and adventitial cells. Staining for fibroblast growth factor receptor 1 revealed a high level of expression in ballooned arteries 3 and 14 days after injury. Taken together, these results provide a molecular and cellular basis for the observed specificity. Prolonged delivery of rFGF2-SAP can affect the natural history of arterial repair after injury.

In Vivo Gene Transfer Into Injured Carotid Arteries

Adenoviral vectors are very attractive agents for use in in vivo arterial gene transfer. In a previous study, we demonstrated high-efficiency adenovirus-mediated gene transfer into medial smooth muscle cells of balloon-injured rat carotid arteries. We now further characterize this system by investigating the reproducibility of recombinant gene expression, the presence of acute adenovirus-associated toxicity in the vessel wall, and the optimal virus concentration for transduction. Balloon-injured rat carotid arteries were incubated with (1) an adenovirus expressing a beta-galactosidase gene (Av1LacZ4), (2) a related adenovirus without the recombinant gene (Addl312), or (3) control solutions. Recombinant gene expression was determined 3 days after gene transfer by measurement of beta-galactosidase activity in vessel extracts and by counting of smooth muscle cells in microscopic sections that were histochemically stained to detect recombinant beta-galactosidase activity. Adenovirus-associated toxicity was assessed in microscopic cross sections by counting of total smooth muscle cell nuclei in the media (to identify cell loss) and characterization of medial cellular infiltrates with histochemical stains for specific inflammatory cells (neutrophils, lymphocytes, macrophages, and monocytes). Maximum recombinant gene expression after incubation with Av1LacZ4 was produced by virus concentrations ranging from 2 x 10(10) to 5 x 10(10) plaque-forming units (pfu)/mL. Surprisingly, use of a higher concentration of Av1LacZ4 virus (1 x 10(11) pfu/mL) resulted in loss of recombinant gene expression. In addition, infusion of either Av1LacZ4 or Addl312 at 1 x 10(11) pfu/mL resulted in statistically significant decreases in medial smooth muscle cell number (53% decrease, P < 0.01 for Av1LacZ4; 39% decrease, P < .05 for Addl312) compared with vessels infused with control solution. This decrease in smooth muscle cell number was not present after the infusion of virus at lower concentrations. The number of neutrophils in vessel cross sections was significantly increased (fivefold; P < .05) after infusion of Av1LacZ4 at 1 x 10(11) pfu/mL compared with vessels infused with control solution. Lymphocytes, macrophages, and monocytes were present only in low numbers in all vessel cross sections and were not increased consequent to adenovirus infusion. This model of focal in vivo adenovirus-mediated gene transfer into the media of injured arteries is highly reproducible and allows high-level recombinant gene expression over a fairly narrow range of virus concentrations. Acute adenovirus-associated tissue toxicity, as demonstrated by medial smooth muscle cell loss and neutrophilic infiltrates, places an upper limit on virus concentration and associated recombinant gene expression and suggests the presence of a "window" of virus concentration in which either therapeutic or biological effects of recombinant genes can be studied in the absence of associated acute toxicity.

Kinetics of bromodeoxyuridine uptake by smooth muscle cells after arterial injury.

The time course of 5-bromo-2'-deoxyuridine (BrdU) uptake by medial and neointimal smooth muscle cells was examined in rat carotid arteries at periods of 1-20 days after balloon catheter injury. DNA-incorporated BrdU was determined by the indirect immunoperoxidase technique in both plastic- and paraffin-embedded specimens. The BrdU labeling index (LI) peaked at 48 h in the media (19.9% paraffin, 9.4% plastic) and at 5 days in the neointima (55.4% paraffin, 60.2% plastic). Immunohistochemistry for the endogenous marker proliferating cell nuclear antigen was variable and generally less reliable than BrdU. Using total viable medial smooth muscle cell number as an index of medial injury, the LI for BrdU was directly proportional to the extent of injury at 48 h, and correlated with loss of actin immunoreactivity. BrdU immunohistochemistry is a reliable alternative to [3H]-thymidine autoradiography for short-term labeling in this model. Proliferation of smooth muscle cells in this model is likely related to the degree of injury to the media.

Hyperplastic growth response of vascular smooth muscle cells following induction of acute hypertension in rats by aortic coarctation.

This study examines the growth response of vascular smooth muscle cells following induction of acute hypertension in rats by partial ligation of the abdominal aorta between the renal arteries. Systolic blood pressures proximal to the ligature increased dramatically within 3 days (from 135 +/- 3 to 195 +/- 7 torr) of surgery while pressures distal to the ligature were reduced from control values. The frequency of smooth muscle cells undergoing DNA replication was increased 25-fold in thoracic aortas of coarctation rats compared to sham-operated controls 9 days post-coarctation, while no differences were observed in cells in abdominal aortic segments 1 cm distal to the ligature. A small but significant increase in the frequency of tetraploid smooth muscle cells was observed in thoracic aortas of coarctation rats, but this increase accounted for less than 2% of the increase in medial DNA content. By far, the major growth response was hyperplasia, as evidenced by a 25% increase in thoracic aortic medial smooth muscle cell number without a change in mean cellular volume (micron3/cell) or mass (ng/cell). Whereas no evidence of endothelial denudation was observed in thoracic aortas of coarctation rats by scanning electron microscopy, endothelial cell turnover rates were increased 23-fold compared to controls, indicating that some form of endothelial "injury" or dysfunction was present. Consistent with this, morphological changes indicative of endothelial injury (e.g., subendothelial edema) were observed by light and transmission electron microscopy. The marked contrast between results of this study and our previous studies showing that aortic medial hypertrophy in spontaneously hypertensive and Goldblatt hypertensive rats was due to cellular hypertrophy and hyperploidy without hyperplasia, clearly demonstrates that the growth response of smooth muscle cells within a given blood vessel can be quite different, depending on the model of hypertension. It is suggested that a non-denuding form of endothelial "injury" may play an important role in the proliferative growth response of smooth muscle cells following induction of coarctation hypertension.