Age increases MCP-1 level in association with bariatric surgery operating time and metabolic risk severity.

OBJECTIVE: Assess the role of inflammation on operating time in younger vs. older bariatric surgery patients. METHODS: Fifty-five younger (F: 46, Age: 34.9 ± 4.0 years, body mass index [BMI]: 48.2 ± 1.0 kg m-2) and 48 older (F: 34, Age: 57.0 ± 5.1 years, BMI: 46.8 ± 1.0 kg m-2) adults were studied prior to surgery. Blood pressure, glycaemic control (fasting glucose/insulin, HbA1c), lipids (high-density lipoprotein and triglycerides) and inflammation (monocyte chemoattractant protein-1 [MCP-1]) were assessed. Metabolic risk severity z-scores were calculated from clinical outcomes. Omental adipose biopsies were collected at surgery for MCP-1 protein analysis. Operating time was used to characterize surgical difficulty. RESULTS: Older vs. younger adults had higher HbA1c (P = 0.03). There was no difference in BMI, lipids, metabolic risk severity or insulin between groups, but operating time was longer in older vs. younger individuals (P = 0.04). Circulating MCP-1 was also elevated in older vs. younger adults (P = 0.04) independent of HbA1c, although this was not explained by omental fat. Nevertheless, serum MCP-1 was associated with increased metabolic risk severity (R = 0.27, P = 0.01). In addition, operating time was linked to HbA1c (R = 0.30, P = 0.01) and omental MCP-1 protein (R = 0.31, P < 0.01). CONCLUSIONS: MCP-1 is associated with longer operating time and increased metabolic risk severity in older bariatric patients independent of glycaemic control. Pre-operative treatment of inflammation may be required to enhance surgery effectiveness.

Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells.

We recently identified hepatoma-derived growth factor (HDGF) as a nuclear targeted vascular smooth muscle cell (VSM) mitogen that is expressed in developing vascular lesions. In the present study, VSM in culture express endogenous HDGF only in the nucleus and target a green fluorescent protein (GFP)-HDGF fusion to the nucleus. To define the features of the HDGF molecule that are essential for nuclear localization and mitogenic function, deletion and site-directed mutagenesis were performed. Deletion analysis identified the carboxyl-terminal half of HDGF to be responsible for nuclear targeting in VSM. Overexpression of tagged HDGF proteins with point mutations in the putative bipartite nuclear localization sequence in the carboxyl terminus demonstrated that single Lys --> Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, the Lys --> Asn mutation of all three lysines blocked nuclear entry. Point mutation of a p34(cdc2) kinase consensus motif within the nuclear localization sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple Lys --> Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that HDGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is required for HDGF stimulation of DNA replication in VSM.

Vascular injury induces posttranscriptional regulation of the Id3 gene: cloning of a novel Id3 isoform expressed during vascular lesion formation in rat and human atherosclerosis.

The molecular mechanisms that regulate the proliferation of smooth muscle cells (SMCs) of the vasculature in response to injury are poorly understood. Members of the inhibitor of DNA binding (Id) class of helix-loop-helix transcription factors are known to regulate the growth of a variety of cell types; however, the expression of the various Id genes in SMCs and in vascular lesions has not been examined. In the present study, the yeast 2-hybrid system was used to clone Id genes from a cultured rat aortic SMC library. By use of ubiquitous E proteins as bait, Id3 and a novel isoform of Id3 (Id3a) were cloned. Id3a is the product of alternative splicing of the Id3 gene, resulting in inclusion of a 115-bp "coding intron," which encodes a unique 29-amino acid carboxyl terminus for the Id3a protein. Unlike Id3, Id3a mRNA was not detected in the normal rat carotid artery. However, after balloon injury, Id3a was abundantly expressed throughout the neointimal layer. In addition, mRNA of the human homologue of Id3a (Id3L) was detected in human carotid atherosclerotic plaques. Adenovirus-mediated overexpression of these Id3 isoforms in cultured rat aortic SMCs revealed that infection of SMCs with an adenovirus overexpressing Id3a (in contrast to Id3) resulted in a significant decrease in cell number versus AdLacZ-infected cells. DNA fragmentation analysis suggested that this decrease in SMC viability was due to increased apoptotic activity in cells infected with adenovirus overexpressing Id3a. These results provide evidence that alternative splicing of the Id3 gene may represent an important mechanism by which neointimal SMC growth is attenuated during vascular lesion formation.

Selective alpha(v)beta(3)-receptor blockade reduces macrophage infiltration and restenosis after balloon angioplasty in the atherosclerotic rabbit.

BACKGROUND: alpha(v)beta(3)-Integrin receptors are upregulated in atherosclerotic arteries and play a key role in smooth muscle cell and possibly inflammatory cell migration. We hypothesized that after balloon angioplasty (BA) of atherosclerotic arteries, selective inhibition of the alpha(v)beta(3)-receptor by XT199, a small-molecule, non-peptide-selective alpha(v)beta(3)-receptor antagonist, would reduce restenosis. METHODS AND RESULTS: After induction of focal atherosclerosis, rabbits underwent femoral BA and received XT199 (2.5 mg/kg IV bolus plus 2.5 mg. kg(-1). d(-1) IV; n=19) or vehicle (n=20) for 14 days. At 28 days after BA, the XT199 group had a larger lumen (0.75+/-0.26 versus 0.57+/-0.20 mm(2), P=0.03) and a smaller neointimal area (0.49+/-0.18 versus 0.68+/-0.25 mm(2), P=0.01) than the vehicle group. Angiographic analysis confirmed a 30% to 40% reduction in restenosis. Arteries harvested at 28 days after BA did not show a reduction in intima plus media smooth muscle cell content but did show a 50% reduction in macrophage cell density in the XT199 group (716+/-452 versus 1458+/-989 cells/mm(2), P<0.006). Neovessel density at 28 days was also reduced (23+/-42 versus 58+/-46 vessel cross sections/mm(2), P<0.02). Early after BA (ie, 3 to 7 days), there was a decrease in intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, indicative of a reduction in vascular cell activation. CONCLUSIONS: Selective alpha(v)beta(3)-receptor blockade for 14 days after BA in the focally atherosclerotic rabbit significantly reduced restenosis and limited macrophage infiltration and neovascularization in the vessel wall.

Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development.

Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8-24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope-tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury.

All-trans-retinoic acid limits restenosis after balloon angioplasty in the focally atherosclerotic rabbit : a favorable effect on vessel remodeling.

All-trans-retinoic acid (atRA) has potent in vitro effects on a number of processes involved in vascular injury and repair, such as modulating smooth muscle cell (SMC) proliferation and inducing SMC differentiation, and may play an important role in the in vivo response to vascular injury. We hypothesized that atRA would limit restenosis after balloon angioplasty through SMC-modulated changes in plaque size and vessel geometry. Balloon angioplasty was performed on rabbits with focal femoral atherosclerosis randomized to treatment with atRA or saline. At 28 days after balloon angioplasty, minimal luminal diameter was significantly larger in the atRA group (1.24+/-0.17 versus 1.12+/-0.22 mm, P=0.02). Histomorphometry confirmed a larger lumen area (0.51+/-0.20 versus 0. 34+/-0.13 mm(2), P=0.004) in the atRA group, with no difference in absolute plaque area. Internal elastic lamina and external elastic lamina areas were significantly larger in the atRA group (0.89+/-0. 27 versus 0.66+/-0.24 mm(2), P=0.001, and 1.29+/-0.38 versus 0. 98+/-0.32 mm(2), P=0.001, respectively). Vessel sections exhibited significantly more alpha-actin and desmin immunostaining (P=0.01) in the atRA-treated group. No differences in early cellular proliferation and collagen content were detected with the use of bromodeoxyuridine. In this atherosclerotic model of vascular injury, atRA limits restenosis after balloon angioplasty by effects secondary to overall vessel segment enlargement at the angioplasty site rather than by effects on plaque size or cellular proliferation. Increased alpha-actin and desmin immunostaining suggest a possible role for phenotypic modulation of SMCs in this favorable remodeling effect.

Early plus delayed hirudin reduces restenosis in the atherosclerotic rabbit more than early administration alone: potential implications for dosing of antithrombin agents.

BACKGROUND: A 2-hour infusion of r-hirudin at the time of balloon angioplasty limits restenosis in atherosclerotic rabbits. Because thrombin activity in the vessel wall after angioplasty remains high for 48 to 72 hours, we hypothesized that a second infusion of hirudin at 24 hours would reduce restenosis more than early treatment alone. METHODS AND RESULTS: Femoral atherosclerosis was induced in 35 rabbits by air desiccation injury and a high-cholesterol diet. At the time of angioplasty, rabbits were randomly assigned to 1 of 4 groups: controls: heparin bolus, saline infusion at 24 hours; early hirudin: hirudin bolus+2 hours' infusion, saline infusion at 24 hours; delayed hirudin: heparin bolus, hirudin infusion+/-bolus at 24 hours; and early+delayed hirudin: hirudin bolus+2 hours' infusion, hirudin infusion+/-bolus at 24 hours. Rabbits were euthanized after 28 days. The early+delayed hirudin treatment group had less loss of minimal lumen diameter by angiography at 28 days. By histomorphometry, cross-sectional area narrowing by plaque was least in the early+delayed treatment group compared with controls (P=0.0001), early hirudin (P=0.01), or delayed hirudin (P=0.001). The early+delayed hirudin group also had a significant reduction in absolute plaque area and an improvement in lumen area compared with the other groups. No differences were observed between treatment groups with respect to the cross-sectional area encompassed by the internal or external elastic laminae. CONCLUSIONS: Combined early+delayed administration of hirudin significantly reduces angiographic restenosis and cross-sectional area narrowing by plaque compared with early or late treatment alone. These results suggest that restenosis after balloon angioplasty is markedly influenced by thrombin-mediated events not only occurring early but also extending beyond the first 24 hours in this model.

Predictors of luminal narrowing by neointima after angioplasty in atherosclerotic rabbits.

OBJECTIVE: The present study was designed to identify the predictors of cross-sectional area narrowing by neointima (%CSAN-N) after balloon angioplasty (BA) in the cholesterol fed rabbit model. METHODS: Angiographic, histomorphometric, and immunohistochemical data were analyzed from 91 femoral arteries of New Zealand white rabbits. Focal atherosclerosis was induced by air desiccation of the endothelium followed by a 2% cholesterol diet for 28 days. The rabbits received heparin (150 U/kg) at the time of BA (2.5 mm; three, 60-second, 10-atm inflations). Arteries were perfusion-fixed and excised 7 (n = 16), 14 (n = 11), 21 (n = 9), or 28 (n = 20) days after BA. Non-angioplastied arteries were de-endothelialized (cholesterol-fed [n = 12] or normal diet [n = 8]), non-injured but cholesterol-fed (n = 7), or normal (n = 8). RESULTS: Univariate regression across all groups showed that the absolute area of the lumen by histomorphometry (LA) correlated significantly with the area bounded by the external elastic lamina (EEL) (vessel size), but no correlation was found with the absolute area of neointima or media, the percentage disruption of the internal elastic lamina (IEL), or the percentage of neointima and media occupied by foam cells. However, %CSAN-N correlated significantly with the area bounded by the EEL, significantly with the absolute neointimal area, and negatively with the absolute LA (p < 0.0001). Significant correlations were also found between %CSAN-N and the % IEL disrupted, the area of neointima and media occupied by RAM-11 + foam cells, and the loss of alpha-actin positivity in the media (p < 0.0001). CONCLUSIONS: These studies show that neointimal formation contributes significantly to luminal narrowing 1 month after angioplasty in this model, that the degree of vascular injury and the extent of foam cell accumulation in the neointima and media are significant independent predictors of neointimal formation, and that the area of the neointima, and the percent narrowing by neointima, are important predictors of remodeling itself (EEL area). These predictors were not identifiable when the analysis was focused on the determinants of absolute luminal area alone.

Effect of thrombin inhibition with desulfatohirudin on early kinetics of cellular proliferation after balloon angioplasty in atherosclerotic rabbits.

BACKGROUND: Thrombin may have a pivotal role in restenosis after angioplasty. Hirudin, a potent thrombin inhibitor, reduces luminal narrowing by plaque after angioplasty in a rabbit model of atherosclerosis. Because cellular proliferation is believed to be an important mechanism for restenosis and thrombin has been shown to be a potent smooth muscle cell mitogen in vitro, we hypothesized that the mechanism of the effect of hirudin on limiting luminal narrowing by plaque occurs via inhibition of cellular proliferation. METHODS AND RESULTS: Femoral atherosclerosis was induced in 108 rabbits, and balloon angioplasty was performed. At angioplasty, group 1 rabbits (n=38) were treated with a 2-hour infusion of hirudin, and group 2 rabbits (n=41) were treated with heparin. Group 3 rabbits (n=29) were treated with hirudin (n=15) or heparin (n=14) and killed at 7 or 28 days to determine cross-sectional area narrowing by plaque and cellular proliferation with the use of bromodeoxyuridine labeling. At 29, 71, or 167 hours after angioplasty, group 1 and 2 rabbits were injected with 3H-thymidine and killed 1 hour later, and labeling indexes were determined. A significant increase in the index of 3H-thymidine-labeled nuclei was observed in the intima of "ballooned" arteries compared with "nonballooned" atherosclerotic arteries at both 30 hours (0.06+/-0.05 versus 0.01+/-0.01, P<.01) and 72 hours (0.10+/-0.06 versus 0.004+/-0.004, P<.01). By 7 days, the index of labeled cells was similar to baseline (0.04+/-0.03 versus 0.01+/-0.01, P=.12). Hirudin had no effect on the 3H-thymidine labeling indexes at any of the time points studied despite the fact that hirudin treatment in group 3 rabbits resulted in less cross-sectional area narrowing by plaque at both 7 and 28 days after angioplasty (41+/-16 versus 24+/-12 at 7 days and 60+/-21 versus 44+/-17 at 28 days, heparin versus hirudin; P<.03). CONCLUSIONS: Balloon angioplasty resulted in a marked increase in cellular proliferation that peaked at 72 hours. A 2-hour infusion of hirudin failed to reduce early 3H-thymidine labeling, suggesting that inhibition of cell proliferation within the first 7 days after angioplasty is not the predominant mechanism by which hirudin exerts its effect on limiting luminal narrowing by plaque 28 days after balloon angioplasty in this animal model.

Thrombin and vascular smooth muscle cell proliferation: implications for atherosclerosis and restenosis.

Despite long-standing knowledge about the relationship between thrombosis and atherosclerosis, the specific role of thrombin in modulating atherosclerosis and the response to vascular injury is not well understood. Thrombin receptor stimulation in vitro signals many cellular events that are associated with the response to vascular injury (atherosclerosis) in vivo. Proliferation of smooth muscle cells (SMCs) is an important component of the response to vascular injury. We have previously shown that human alpha-thrombin and the 14-amino acid human thrombin receptor-activating peptide (huTRAP-14) stimulate proliferation of cultured rat aortic SMCs. However, thrombin-induced SMC proliferation demonstrates delayed kinetics relative to platelet-derived growth factor (PDGF-BB, another potent SMC mitogen). Several mechanisms may be responsible for these delayed kinetics in vitro, including production of necessary secondary growth factors and thrombin-induced upregulation of its receptor. In vivo studies have demonstrated that thrombin inhibition limits the response to vascular injury in a hypercholesterolemic rabbit model of focal femoral atherosclerosis. However, this effect does not appear to be mediated by effects on early SMC proliferation. In this discussion, we will address the mechanisms of thrombin-induced SMC proliferation in vitro and apply this knowledge to our understanding of the role of thrombin inhibition in limiting the response to vascular injury in vivo.

Thrombin-induced mitogenesis in cultured aortic smooth muscle cells requires prolonged thrombin exposure.

Thrombin has been implicated in vascular smooth muscle cell (VSMC) proliferation after vessel injury. Its proliferative effects, which are mediated via proteolytic activation of a receptor similar or identical to the cloned thrombin receptor (TR), have markedly delayed kinetics. The present study demonstrates that, despite rapid thrombin receptor activation and similar time to S phase entry compared with classic polypeptide growth factors, prolonged thrombin exposure is required to promote maximal VSMC mitogenesis. Flow cytometric analysis of thrombin-stimulated cells revealed that thrombin induced a progressive increase in the growth fraction over 3 days in culture, an effect that was blocked by hirudin even late after thrombin addition. Northern blot hybridization after thrombin stimulation demonstrated that thrombin upregulates TR mRNA expression within 6 h. These findings indicate that VSMC proliferate in response to prolonged thrombin exposure and suggest that the mitogenic delay may involve not only the thrombin-dependent synthesis and activation of newly made TR but also the progressive thrombin-dependent recruitment of cells into the growth fraction.

Nuclear proteins bind a cis-acting element in the smooth muscle alpha-actin promoter.

Identification of the regulators of smooth muscle cell (SMC) gene expression is critical to understanding SMC differentiation and alterations in SMC phenotype in vascular disease. Previous studies revealed positive transcriptional activity within the chicken smooth muscle (SM) alpha-actin promoter region from -209 to -257. In the present study, transient transfections of wild-type and mutant chicken SM alpha-actin promoter/reporter gene constructs into rat aortic SMC demonstrated that the positive transcriptional activity of this region was abolished with a two base pair mutation in a conserved sequence motif at -225 to -233 (TGTTTATC to TACTTATC). Electrophoretic mobility shift assays revealed that nuclear factors bound promoter fragments containing this sequence and that specific mutations in the TGTTTATC motif abolished nuclear factor binding. Studies thus provide evidence for binding of a nuclear factor to a positive cis-acting element within the SM alpha-actin promoter. Further characterization of this factor may contribute to a better understanding of the molecular mechanisms that regulate differentiation of SMC in vascular disease.

Thrombin-induced mitogenesis of vascular SMC is partially mediated by autocrine production of PDGF-AA.

Previous studies have shown that alpha-thrombin (Thr) is a mitogen for cultured smooth muscle cells (SMC), but controversy exists concerning mechanisms of growth stimulation. The purpose of these studies was to determine whether autocrine production of platelet-derived growth factor-AA (PDGF-AA) mediated Thr-induced SMC mitogenesis. SMC derived from aortae of Sprague-Dawley rats were grown to confluence, growth arrested in serum-free medium, and treated. Conditioned medium (CM) was harvested by adding bovine serum albumin as a carrier and hirudin to neutralize residual Thr. Results demonstrated that CM from Thr-treated SMC (Thr CM) had increased mitogenic activity compared with control CM (Cnt CM) and that PDGF-AA levels were increased 12-fold in Thr CM compared with Cnt CM. The excess mitogenic activity in Thr CM was markedly inhibited by PDGF-AA-neutralizing antibodies. Cotreatment with Thr and PDGF-AA-neutralizing antibodies resulted in a 30% decrease in Thr-induced [3H]thymidine incorporation. These results demonstrate that autocrine production of PDGF-AA partially mediated Thr-induced SMC mitogenesis.

Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor.

Thrombin has been implicated in the stimulation of smooth muscle cell (SMC) proliferation that contributes to post angioplasty restenosis. The present studies demonstrated that human alpha-thrombin was a potent and efficacious mitogen for cultured rat aortic SMC, stimulating an increase in 3H-thymidine incorporation, as well as an increase in cell number at 1 to 10 nM concentration. gamma-Thrombin, which is enzymatically active but lacks fibrinogen clotting activity, stimulated SMC mitogenesis but was approximately 10-fold less potent than alpha-thrombin. In contrast, D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone-alpha-thrombin, which lacked enzymatic activity, had no mitogenic effect. Diisopropylfluorophosphate-alpha-thrombin failed to stimulate mitogenesis except at concentrations having equivalent enzymatic activity as that of alpha-thrombin at its threshold for mitogenesis. Thus, thrombin-induced proliferation was dependent on enzymatic activity. A 14-residue peptide (SFLLRNPNDKYEPF) corresponding to amino acids 42 through 55 of the human thrombin receptor (Vu, T. K., D. T. Hung, V. I. Wheaton, and S. R. Coughlin, 1991. Cell. 64:1057-1068) had full efficacy in stimulating SMC proliferation. Reversing the first two amino acids of this peptide abolished mitogenic activity. Northern analysis demonstrated that SMC expressed a single mRNA species that hybridized to a labeled thrombin receptor cDNA probe. These findings indicate that alpha-thrombin stimulates SMC proliferation via the proteolytic activation of a receptor very similar or identical to that previously identified.

Comparison of thrombolytic therapy for acute myocardial infarction in rural and urban settings.

In this study, a tertiary care hospital served as a registry and information source to rural hospitals in northwestern Ohio where thrombolytic therapy had not previously been used. The study was designed to compare the safety and efficacy of intravenous thrombolytic therapy for acute myocardial infarction in the two settings. Fifty-five patients in eight rural hospitals and 36 patients in the urban tertiary care center received intravenous streptokinase. Of the 87 patients whose symptoms first occurred out of the hospital, 63 percent were treated within three hours. There were no significant differences in rates of clinically determined coronary artery recanalization (63 percent versus 69 percent for rural and tertiary hospitals, respectively), in-hospital mortality (5.4 percent versus 11 percent), bleeding complications (3.6 percent versus 5.5 percent), or time from the onset of pain to infusion of streptokinase (3.4 hours versus 2.9 hours). There were also no differences in the completeness of collection of serial coagulation data and cardiac enzyme values, or in the documentation of chest pain onset and cessation. Major differences between rural centers and the tertiary care center involved the use of serial electrocardiography (58 percent versus 89 percent, respectively), subsequent cardiac catheterization (49 percent versus 86 percent), and the timing of catheterization, when performed (30.4 days versus 4.6 days) (p less than 0.005 for all values). Thrombolytic therapy for acute myocardial infarction can be administered quickly, safely, and effectively in rural hospital settings even by physicians previously unfamiliar with this form of treatment.