The molecular mechanism of actinomycin D in preventing neointimal formation in rat carotid arteries after balloon injury

Summary The pathological mechanism of restenosis is primarily attributed to excessive proliferation of vascular smooth muscle cells (SMC). Actinomycin D has been regarded as a potential candidate to prevent balloon injury-induced neointimal formation. To explore its molecular mechanism in regulating cell proliferation, we first showed that actinomycin D markedly reduced the SMC proliferation via the inhibition of BrdU incorporation at 80 nM. This was further supported by the G1-phase arrest using a flowcytometric analysis. Actinomycin D was extremely potent with an inhibitory concentration IC₅₀ at 0.4 nM, whereas the lethal dose LD₅₀ was at 260 μM. In an in vivo study, the pluronic gel containing 80 nM and 80 μM actinomycin D was applied topically to surround the rat carotid adventitia; the thickness of neointima was substantially reduced (45 and 55%, respectively). The protein expression levels of proliferating cell nuclear antigen (PCNA), focal adhesion kinase (FAK), and Raf were all suppressed by actinomycin D. Extracellular signal-regulated kinases (Erk) involved in cell-cycle arrest were found to increase by actinomycin D. These observations provide a detailed mechanism of actinomycin D in preventing cell proliferation thus as a potential intervention for restenosis.     

Quantitative comparison of three rat models of Achilles tendon injury: A multidisciplinary approach

The Achilles tendon, while the strongest and largest tendon in the body, is frequently injured. Inconclusive evidence exists regarding treatment strategies for both complete tears and partial tears. Well-characterized animal models of tendon injury are important for understanding physiological processes of tendon repair and testing potential therapeutics. Utilizing three distinct models of rat Achilles tendon injury, the objective of this study was to define and compare the effects and relative impact on tendon properties and ankle function of both tear severity (complete tear versus partial tear, both with post-operative immobilization) and immobilization after partial tear (partial tear with versus without immobilization). We hypothesized that a complete tear would cause inferior post-injury properties compared to a partial tear, and that immediate loading after partial tear would improve post-injury properties compared to immobilization. All models were reproducible and had distinct effects on measured parameters. Injury severity drastically influenced tendon healing, with complete tear causing decreased ankle mobility and tendon mechanics compared to partial tears. One week of plantarflexion immobilization had a strong effect on animals receiving a partial tear. Tendons with partial tears and immobilization failed early during fatigue cycling three weeks post-injury. Partial tear without immobilization had no effect on ankle range of motion through dorsiflexion at any time point compared to the pre-surgery value, while partial tear with immobilization demonstrated diminished function at all post-injury time points. All three models of Achilles injury could be useful for tendon healing investigations, chosen based on the prospective applications of a potential therapeutic.     

A physico-chemical comparison of aortic receptors in rat hypertension models

Rat models of genetic hypertension include spontaneous hypertension and resistance or sensitivity to mineralocorticoid and salt induced hypertension. Previously, altered aldosterone binding to corticoid receptor I was found in aortic smooth muscle cells cultured from Fischer 344 rats which are extremely resistant to steroid and salt induced hypertension. The corticoid receptor I of Fisher 344 rats had a lower affinity than that of salt sensitive Wistar-Kyoto controls, as well as spontaneously hypertensive rats and Sprague-Dawley rats. In the present study, we have used DEAE-cellulose ion exchange chromatography to compare the structure (charge properties) and steroid specificity of vascular corticoid receptor I and II sites in these same rat hypertension models. No variations in ion exchange properties of type I and II receptors were found. Together with the lower aldosterone affinity of corticoid receptor I sites in Fischer 344 rats these data suggest an altered binding domain which is not seen as a difference in charge density of the receptor protein by ion exchange chromatography.     

Expression of T-cadherin in the rat carotid artery wall after balloon injury and in different rat organs

T-cadherin is an unusual glycosilphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. In contrast to classical cadherins, tissue distribution of T-cadherin so far remained unknown. We examined tissue distribution of T-cadherin in rats using Western blotting and immunohistochemical method. Our results show that T-cadherin is expressed in all types of muscles (cardiac, striated, and smooth muscles), in brain neurons, and spinal cord, in the vessel endothelium, at the apical pole of intestinal villar epithelium, in the basal layer of skin, and eosophagal epithelium. Blood-derived and lymphoid cells as well as connective tissue were T-cadherin-negative. The highest level of T-cadherin expression was revealed in the cardiovascular system. Although T-cadherin was detected in smooth muscle cells, its role in the intimal thickening and restenosis is not known. We examined T-cadherin expression within 1-28 days after balloon injury of rat left carotid arteries. T-cadherin expression was valued immunohistochemically with semiquantitative method. In uninjured arteries, T-cadherin was expressed in endothelial (vWF-positive) cells, and smooth muscle (alpha-actin-positive) cells (SMCs). After denudation of arterial wall, T-cadherin was present both in the media and neointima. We revealed dynamics of T-cadherin expression in the media of injured artery: an essential increase being registered at the stage of cell migration and proliferation in the media and neointima (1-7 days), followed by its decrease to the baseline level (10-28 days). The high upregulation of T-cadherin expression in the media and neointima during migration and proliferation of vascular cells after vessel injury enables us to suggest the involvement of T-cadherin in vessel remodeling after balloon catheter injury.     

The comparison of immobility time in experimental rat swimming models

Rat swimming models have been used in studies about stress and depression. However, there is no consensus about interpreting immobility (helplessness or adaptation) in the literature. In the present study, immobility time, glucose and glycogen mobilization, corticosterone and the effect of desipramine and diazepam were investigated in two different models: swimming stress and the forced swimming test. Immobility time was lower in swimming stress than in the forced swimming test. Both swimming models increased corticosterone levels in comparison with control animal levels. Moreover, swimming stress induced higher corticosterone levels than the forced swimming test did [F(2,14)=59.52; p<0.001]. Liver glycogen content values differed from one another (swimming stress相似文献   

Nitrotyrosine formation and apoptosis in rat models of ocular injury

This study was performed to examine inducible nitric oxide synthase (NOS-2) expression, nitrotyrosine formation and apoptosis in rats with elevated intraocular pressure (IOP) and/or ocular inflammation. Ocular inflammation was induced via injection of intra-vitreal lipopolysaccharide (LPS) while IOP was elevated by episcleral vessel cauterization. Animals were randomized to one of the following conditions: elevated IOP, LPS, elevated IOP+LPS, and control. Immunohistochemical staining and western blot analysis of retinal lysates revealed NOS-2 and nitrotyrosine immunoreactivity in all disease groups. NOS-2 expression and protein nitration was significantly greater in rats with elevated IOP+LPS compared to elevated IOP, LPS, and control groups. Nitrite levels in the retina affirmed significantly increased levels of nitric oxide generation in LPS-treated rats with elevated IOP (346 ± 23.8 μM) vs LPS-treated, elevated IOP and control groups (195.6 ± 12.6, 130 ± 2.5 and 76.6 ± 15.6 μM, respectively). Retinal TUNEL staining showed apoptosis in all diseased groups. Percent of apoptotic cells was significantly greater in the elevated IOP+LPS group compared to LPS-treated or elevated IOP groups. Presented data illustrates that both elevated IOP and ocular inflammation augment NOS-2 expression, retinal protein nitration and apoptosis in rats.     

Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.     

Upregulation of proteinase-activated receptors and hypercontractile responses precede development of arterial lesions after balloon injury

Thrombin and other proteinases exert vascular effects by activating the proteinase-activated receptors (PARs). The expression of PARs has been shown to be upregulated after balloon injury and in human arteriosclerosis. However, the relationship between the receptor upregulation and the alteration of vasomotor function remains to be elucidated. We herein demonstrated that the contractile responses to the PAR-1 and PAR-2 agonist were markedly enhanced in the rabbit femoral arteries after balloon injury. Neointimal thickening was established 4 wk after the injury. No histological change was observed in the sham operation, where the saphenous artery was ligated without any balloon injury. The contractile response to K(+) depolarization was significantly attenuated 1 wk after the injury and then partly recovered after 4 wk. Thrombin, PAR-1-activating peptide, trypsin, and PAR-2-activating peptide induced no significant contraction in the control. All these stimulants induced enhanced responses 1 wk after balloon injury. Such enhanced responses were seen 4 wk after the injury, except for thrombin. There was no change in the Ca(2+) sensitivity of the contractile apparatus as evaluated in the permeabilized preparations. PAR-1-activating peptide (100 mumol/l), but no other stimulants, induced an enhanced contraction in the sham operation. The expression of PAR-1 and PAR-2 slightly increased after the sham operation, whereas it markedly and significantly increased after balloon injury. Our observations suggest that balloon injury induced the receptor upregulation, thereby enhancing the contractile response before the establishment of vascular lesions. The local inflammation associated with the sham operation may also contribute to the receptor upregulation.     

Induction of gicerin/CD146 in the rat carotid artery after balloon injury

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It is reported that the human homologous molecule, CD146, is expressed in the endothelial cells. Here, we found that the expression of gicerin was increased in the rat carotid arteries after balloon injury. Immunohistochemical analysis demonstrated that the expression of gicerin protein was increased in the medial smooth muscle cells prior to the formation of neointima one week after the injury and was also increased in the luminal edge of the neointima after two weeks. We employed A10 cells, a cell line derived from rat aortic smooth muscle cell, and examined the effect of growth factors on the expression of gicerin, such as IGF-1, PDGF-BB, and bFGF. We found that IGF-1, but not PDGF-BB and bFGF, significantly increases the expression of gicerin protein in A10 cells. These suggest gicerin might be involved in the arteriosclerotic neointima formation in the artery.     

Losartan, a nonpeptide angiotensin II (Ang II) receptor antagonist, inhibits neointima formation following balloon injury to rat carotid arteries.

Angiotensin-converting enzyme inhibitors have been shown to inhibit intimal thickening following balloon catheterization of rat carotid arteries. To assess the role of the renin-angiotensin pathway and the angiotensin type-I (AT1) receptor in this effect, the nonpeptide Ang II antagonist losartan (DuP 753) or vehicle was infused continuously i.v. in rats from two days before to two weeks after balloon injury to the left common carotid artery; drug effects upon intimal thickening were examined histologically. Losartan produced a dose-dependent reduction in cross-sectional area of intimal lesions determined two weeks post balloon injury. At 5 mg/kg/day a nonsignificant 23% reduction of intimal area was observed. At the higher dose of 15 mg/kg/day, losartan produced a 48% reduction in intimal area (P less than 0.05) compared to the vehicle-infused group. The cellular density of the neointima was not affected by losartan, indicating a probable effect of the drug upon migration and/or proliferation of smooth muscle cells. In separate groups of non-ballooned rats, losartan infusions of 5 and 15 mg/kg/day produced significant rightward shifts (averaging 6.4- and 55-fold, respectively) in curves relating increases in blood pressure to intravenous Ang II in pithed rats determined between 2 and 16 days following initiation of losartan infusion. Mean arterial blood pressure (determined under alpha-chloralose anesthesia) was reduced following continuous losartan infusion for 6 days from 128 +/- 8 mm Hg (vehicle) to 105 +/- 8 mm Hg at 5 mg/kg/day (P less than 0.05), and 106 +/- 4 mm Hg at 15 mg/kg/day (P less than 0.05). Thus, losartan attenuated the vascular response to balloon catheter injury, and this effect was associated with functional block of vascular AT1 receptors. The results support a role for Ang II, acting via AT1 receptors, in myointimal thickening subsequent to balloon injury of rat carotid arteries.     

Microglial activation in different models of peripheral nerve injury of the rat

Pain and pain modulation has been viewed as being mediated entirely by neurons. However, new research implicates spinal cord glia as key players in the creation and maintenance of pathological pain. Sciatic nerve lesions are one of the most commonly studied pain-related injuries. In our study we aimed to characterize changes in microglial activation in the rat spinal cord after axotomy and chronic constriction injury of the sciatic nerve and to evaluate this activation in regard to pain behavior in injured and control groups of rats. Microglial activation was observed at ipsilateral side of lumbar spinal cord in all experimental groups. There were slight differences in the level and extent of microglial activation between nerve injury models used, however, differences were clear between nerve-injured and sham animals in accordance with different level of pain behavior in these groups. It is known that activated microglia release various chemical mediators that can excite pain-responsive neurons. Robust microglial activation observed in present study could therefore contribute to pathological pain states observed following nerve injury.     

Atherosclerotic lesions of supra-aortic arteries in diabetic patients

The aim of this prospective study was to determine the prevalence and localization of stenotic atherosclerotic lesions of supra-aortic arteries in diabetic patients according to age and sex. Angiograms obtained by digital subtraction angiography were analyzed in 150 diabetic patients (study group) and 150 non-diabetic patients (control group) with symptoms of cerebral ischemia. Diabetic patients were found to have a significantly higher prevalence of stenotic atherosclerotic lesions of the internal carotid artery. Lesions of the large supra-aortic arteries were significantly more common in the left than in the right side of the neck (p < 0.001), but the difference between the diabetic and the non-diabetic group did not reach statistical significance. Hemodynamic conditions were found to be more important than diabetes for the occurrence of atherosclerotic lesions in these arteries. Changes in the proximal segment of the left common carotid artery were the most common finding in diabetic patients, hence attention should be paid to this localization on control examinations.     

Immunolocalization of matrix metalloproteinases in rabbit carotid arteries after balloon denudation

 Extracellular matrix-degrading enzymes may play a key role in vascular remodeling after arterial wall injury. We investigated the immunolocalization of matrix metalloproteinases (MMPs) in rabbit carotid arteries after balloon denudation. Positive immunostaining for MMP-1, -2, -3, and -9 appeared through the neointima 1 week after balloon denudation. The localization of immunopositive smooth muscle cells (SMCs) for MMP-1, -3, and -9, particularly for MMP-9, was almost similar to that of replicative SMCs and became confined to the luminal surface layer of the neointima at later time periods. However, MMP-2-positive SMCs appeared also in the basal layer of the neointima at 2 weeks, increased at 4 weeks, and then totally occupied the neointima at 6 weeks. The MMP-2-positive SMCs in the basal layer of the neointima at 4 and 6 weeks were negative for proliferation-associated antigens and were surrounded by extracellular matrix proteins. Our results suggest that all MMPs act in coordination to promote replication and migration of SMCs in the earlier phases of neointimal formation and that MMP-2 independently contributes to the later stages by facilitating the migration but not replication of SMCs from the media to the intima. Accepted: 25 June 1997