Acute impact of intermittent pneumatic leg compression frequency on limb hemodynamics, vascular function, and skeletal muscle gene expression in humans

The mechanisms by which intermittent pneumatic leg compression (IPC) treatment effectively treats symptoms associated with peripheral artery disease remain speculative. With the aim of gaining mechanistic insight into IPC treatment, the purpose of this study was to investigate the effect of IPC frequency on limb hemodynamics, vascular function, and skeletal muscle gene expression. In this two study investigation, healthy male subjects underwent an hour of either high-frequency (HF; 2-s inflation/3-s deflation) or low-frequency (LF; 4-s inflation/16-s deflation) IPC treatment of the foot and calf. In study 1 (n = 11; 23.5 ± 4.7 yr), subjects underwent both HF and LF treatment on separate days. Doppler/ultrasonography was used to measure popliteal artery diameter and blood velocity at baseline and during IPC treatment. Flow-mediated dilation (FMD) and peak reactive hyperemia blood flow (RHBF) were determined before and after IPC treatment. In study 2 (n = 19; 22.0 ± 4.6 yr), skeletal muscle biopsies were taken from the lateral gastrocnemius of the treated and control limb at baseline and at 30- and 150-min posttreatment. Quantitative PCR was used to assess mRNA concentrations of genes associated with inflammation and vascular remodeling. No treatment effect on vascular function was observed. Cuff deflation resulted in increased blood flow (BF) and shear rate (SR) in both treatments at the onset of treatment compared with baseline (P < 0.01). BF and SR significantly diminished by 45 min of HF treatment only (P < 0.01). Both treatments reduced BF and SR and elevated oscillatory shear index compared with baseline (P < 0.01) during cuff inflation. IPC decreased the mRNA expression of cysteine-rich protein 61 from baseline and controls (P <0 .01) and connective tissue growth factor from baseline (P < 0.05) in a frequency-dependent manner. In conclusion, a single session of IPC acutely impacts limb hemodynamics and skeletal muscle gene expression in a frequency-dependent manner but does not impact vascular function.     

Intermittent pneumatic leg compressions enhance muscle performance and blood flow in a model of peripheral arterial insufficiency

Despite the escalating prevalence in the aging population, few therapeutic options exist to treat patients with peripheral arterial disease. Application of intermittent pneumatic leg compressions (IPC) is regarded as a promising noninvasive approach to treat this condition, but the clinical efficacy, as well the mechanistic basis of action of this therapy, remain poorly defined. We tested the hypothesis that 2 wk of daily application of IPC enhances exercise tolerance by improving blood flow and promoting angiogenesis in skeletal muscle in a model of peripheral arterial insufficiency. Male Sprague-Dawley rats were subjected to bilateral ligation of the femoral artery and randomly allocated to treatment or sham groups. Animals were anesthetized daily and exposed to 1-h sessions of bilateral IPC or sham treatment for 14-16 consecutive days. A third group of nonligated rats was also studied. Marked increases in treadmill exercise tolerance (～33%, P < 0.05) and improved muscle performance in situ (～10%, P < 0.05) were observed in IPC-treated animals. Compared with sham-treated controls, blood flow measured with isotope-labeled microspheres during in situ contractions tended to be higher in IPC-treated animals in muscles composed of predominantly fast-twitch white fibers, such as the plantaris (～93%, P = 0.02). Capillary contacts per fiber and citrate synthase activity were not significantly altered by IPC treatment. Collectively, these data indicate that IPC improves exercise tolerance in a model of peripheral arterial insufficiency in part by enhancing blood flow to collateral-dependent tissues.     

Modulation of collateral artery growth in a porcine hindlimb ligation model using MCP-1

For an appropriate extrapolation to patients with peripheral arterial obstructive disease, we tested the efficacy of monocyte chemoattractant protein 1 (MCP-1) treatment in a porcine hindlimb ligation model. In 40 minipigs, a femoral artery ligation was performed. Control animals were examined immediately after ligation (n = 4) or after 2 wk of intra-arterial infusion of PBS (n = 11). A second group of animals was evaluated after intra-arterial infusion of 2.0 microg/h of MCP-1 for 48 h (followed by 12 days of PBS; n = 13) or 2 wk continuously (n = 12). In the terminal experiment after 2 wk, resting flow to the leg and peripheral arterial pressures were assessed without vasodilatation. Subsequently, vascular conductance was determined by using a pump-driven extracorporal circulation during maximal vasodilatation. The results showed that resting blood flow to the hindlimb was 53% of the normal after 2 wk of infusion of PBS, compared with 81% in both MCP-1 treatment groups (P < 0.05). Collateral conductance was 645 +/- 346 ml x min(-1) x mmHg(-1) after 2 wk of infusion with PBS, compared with 1,070 +/- 530 and 1,158 +/- 535 ml x min(-1) x mmHg(-1) after 48 h and 2 wk treatment with MCP-1, respectively (P < 0.05). Modulation of the process of arteriogenesis is feasible in this large animal model via intra-arterial infusion of the Cys-Cys-chemokine MCP-1.     

Local gene silencing of monocyte chemoattractant protein-1 prevents vulnerable plaque disruption in apolipoprotein E-knockout mice

Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine (CCL2), has been demonstrated to play important roles in atherosclerosis and becoming an important therapeutic target for atherosclerosis. The present study was undertaken to test the hypothesis that local RNAi of MCP-1 by site-specific delivery of adenovirus-mediated small hairpin RNA (shRNA) may enhance plaque stability and prevent plaque disruption in ApoE-/- mice. We designed an adenovirus-mediated shRNA against mouse MCP-1 (rAd5-MCP-1-shRNA). Male apolipoprotein E-knockout (ApoE-/-) mice (n = 120) were fed a high-fat diet and vulnerable plaques were induced by perivascular placement of constrictive collars around the carotid artery, intraperitoneal injection of lipopolysaccharide and stress stimulation. Mice were randomly divided into RNA interference (Ad-MCP-1i) group receiving local treatment of rAd5-MCP-1-shRNA suspension, Ad-EGFP group receiving treatment of rAd5-mediated negative shRNA and mock group receiving treatment of saline. Two weeks after treatment, plaque disruption rates were significantly lower in the Ad-MCP-1i group than in the Ad-EGFP group (13.3% vs. 60.0%, P = 0.01), and local MCP-1 expression was significantly inhibited in the Ad-MCP-1i group confirmed by immunostaining, qRT-PCR and western blot (P<0.001). Compared with the Ad-EGFP group, carotid plaques in the Ad-MCP-1i group showed increased levels of collagen and smooth muscle cells, and decreased levels of lipid and macrophages. The expression of inflammatory cytokines and activities of matrix metalloproteinases (MMPs) were lower in the Ad-MCP-1i group than in the Ad-EGFP group. In conclusion, site-specific delivery of adenoviral-mediated shRNA targeting mouse MCP-1 downregulated MCP-1 expression, turned a vulnerable plaque into a more stable plaque phenotype and prevented plaque disruption. A marked suppression of the local inflammatory cytokine expression may be the central mechanism involved.     

VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise-trained rats

Both collateral vessel enlargement (arteriogenesis) and capillary growth (angiogenesis) in skeletal muscle occur in response to exercise training. Vascular endothelial growth factor (VEGF) is implicated in both processes. Thus we examined the effect of a VEGF receptor (VEGF-R) inhibitor (ZD4190, AstraZeneca) on collateral-dependent blood flow in vivo and collateral artery size ex vivo (indicators of arteriogenesis) and capillary contacts per fiber (CCF; an index of angiogenesis) in skeletal muscle of both sedentary and exercise-trained rats 14 days after bilateral occlusion of the femoral arteries. Total daily treadmill run time increased appreciably from approximately 70 to approximately 100 min (at 15-20 m/min, twice per day) and produced a large (approximately 75%, P < 0.01) increase in calf muscle blood flow and a greater size of the collateral artery (wall cross-sectional area). ZD4190, which previously has been shown to inhibit the activity of VEGF-R2 and -R1 tyrosine kinase in vitro (IC50 = 30 and 700 nM, respectively), completely blocked the increase in collateral-dependent blood flow and inhibited collateral vessel enlargement. Thus exercise-stimulated collateral arteriogenesis appears to be completely dependent on VEGF-R signaling. Interestingly, enhanced mRNA expression of the VEGF family ligand placental growth factor (2- to 3.5-fold), VEGF-R1 (approximately 2-fold), and endothelial nitric oxide synthase (2- to 3.5-fold) in an isolated collateral artery implicates these factors as important in arteriogenesis. Training of ischemic muscle also induced angiogenesis, as shown by an increase (approximately 25%, P < 0.01) in CCF in white gastrocnemius muscle. VEGF-R inhibition only partially blocked (P < 0.01) but did not eliminate the increase (P < 0.01) in capillarity. Our findings indicate that VEGF-R tyrosine kinase activity is essential for collateral arteriogenesis and important for the angiogenesis induced in ischemic muscle by exercise training; however, other angiogenic stimuli are also important for angiogenesis in flow-limited active muscle.     

Role of nitric oxide in vasodilation in upstream muscle during intermittent pneumatic compression.

This study investigated the dosage effects of nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on intermittent pneumatic compression (IPC)-induced vasodilation in uncompressed upstream muscle and the effects of IPC on endothelial NOS (eNOS) expression in upstream muscle. After L-NMMA infusion, mean arterial pressure increased by 5% from baseline (99.5 +/- 18.7 mmHg; P < 0.05). Heart rate and respiratory rate were not significantly affected. One-hour IPC application on legs induced a 10% dilation from baseline in 10- to 20-microm arterioles and a 10-20% dilation in 21- to 40 microm arterioles and 41- to 70-microm arteries in uncompressed cremaster muscle. IPC-induced vasodilation was dose dependently reduced, abolished, or even reversed by concurrently infused L-NMMA. Moreover, expression of eNOS mRNA in uncompressed cremaster muscle was upregulated to 2 and 2.5 times normal at the end of 1- and 5-h IPC on legs, respectively, and the expression of eNOS protein was upregulated to 1.8 times normal. These increases returned to baseline level after cessation of IPC. The results suggest that eNOS plays an important role in regulating the microcirculation in upstream muscle during IPC.     

Anti-tumor necrosis factor-{alpha} therapies attenuate adaptive arteriogenesis in the rabbit

The specific antagonists of tumor necrosis factor-alpha (TNF-alpha), infliximab and etanercept, are established therapeutic agents for inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Although the importance of TNF-alpha in chronic inflammatory diseases is well established, little is known about its implications in the cardiovascular system. Because proliferation of arteriolar connections toward functional collateral arteries (arteriogenesis) is an inflammatory-like process, we tested in vivo the hypothesis that infliximab and etanercept have antiarteriogenic actions. Sixty-three New Zealand White rabbits underwent femoral artery occlusion and received infliximab, etanercept, or vehicle according to clinical dosage regimes. After 1 wk, collateral conductance, assessed with fluorescent microspheres, revealed significant inhibition of arteriogenesis (collateral conductance): 52.4 (SD 8.1), 35.2 (SD 7.7), and 33.3 (SD 10.1) ml x min(-1) x 100 mmHg(-1) with PBS, infliximab, and etanercept, respectively (P < 0.001). High-resolution angiography showed no significant differences in number of collateral arteries, but immunohistochemical analysis demonstrated a decrease in mean collateral diameter, proliferation of vascular smooth muscle cells, and reduction of leukocyte accumulation around collateral arteries in treated groups. Infliximab and etanercept bound to infiltrating leukocytes, which are important mediators of arteriogenesis. Infliximab induced monocyte apoptosis, and neither substance affected monocyte expression of the adhesion molecule Mac-1. We demonstrated that TNF-alpha serves as a pivotal modulator of arteriogenesis, which is attenuated by treatment with TNF-alpha inhibitors. Reduction of collateral conductance is most likely due to inhibition of perivascular leukocyte infiltration and subsequent lower vascular smooth muscle cell proliferation. This is the first report showing a negative influence of TNF-alpha inhibitors on collateral artery growth.     

Protein expression pattern in experimental pneumococcal meningitis

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.     

Expression of MCP-1 in the Hippocampus of SHRSP with Ischemia-Related Delayed Neuronal Death

1. The expression of monocyte chemoattractant protein-1 (MCP-1) was examined in stroke-prone spontaneously hypertensive rats with transient global ischemia in order to study the involvement of the infiltration of blood monocytes in the mechanism of ischemia-related neuronal death.2. The brains of the animals with occlusion of the bilateral carotid arteries for 10 min were removed at 8 h, 1, 2, 4 and 7 days after reperfusion. Frozen sections were used for in situ hybridization and tissue specimens from the hippocampus and the cerebral cortex were used to measure the concentration of MCP-1 by ELISA.3. No MCP-1 mRNA was detected in the hippocampus of the sham group animals. One day after ischemia-reperfusion, MCP-1 mRNA was clearly expressed in the CA4 subfield and the molecular layer of the dentate gyrus, while it was slightly expressed in the lacnosum moleculare of the CA1 subfield. A dramatic expression was demonstrated in the entire CA1 subfield at 2 days after the operation. Most of the cells expressing MCP-1 were astrocytes. At 4 and 7 days after reperfusion, no MCP-1 mRNA was detected in the hippocampus. The concentration of MCP-1 protein dramatically increased in the hippocampus at 2 days after reperfusion.4. Taken together with the findings of our previous study showing an increased permeability of the blood-brain barrier in the hippocampus from 12 h after ischemia-reperfusion, the astrocytes expressing MCP-1 might therefore induce the migration of monocytes into the brain parenchyma. As a result, such astrocytes expressing MCP-1 may therefore be related to the pathological events of delayed neuronal death in the pyramidal neurons.     

C-reactive protein correlates with macrophage accumulation in coronary arteries of hypercholesterolemic pigs.

A growing body of evidence supports the hypothesis that C-reactive protein (CRP) is a marker of inflammation in coronary artery disease. The purpose of the present study was to test the hypothesis that CRP correlates with macrophage accumulation during the initial stages of coronary vascular disease. Adult male pigs were fed a normal chow (NF) or a high-fat high-cholesterol (HF) diet for 20 wk. After 20 wk, blood was collected for analyses of interleukin-6 (IL-6), CRP, and lipids. After blood collection, the pigs were euthanized and the right coronary arteries (RCA) were harvested and fixed in neutral buffered formalin. Paraffin-embedded sections of RCA were stained immunohistochemically for CRP, scavenger receptor A (SRA), and monocyte chemoattractant protein-1 (MCP-1). All cholesterol fractions were elevated in the HF vs. the NF group (P < 0.05). There was little or no positive staining for CRP, SRA, or MCP-1 in the RCA of NF pigs, but there was extensive staining in lipidladen macrophage foam cells in the HF pigs. Double staining revealed colocalization of CRP with SRA and CRP with MCP-1 in foam cells. Serum IL-6 was below the assay detection limit in all pigs. Serum CRP correlated directly with plasma total cholesterol (R = 0.727, P = 0.041) and accumulation of SRA-positive macrophages (R = 0.938, P < 0.001) in RCA of HF pigs. We conclude that serum CRP correlates with macrophage accumulation and coronary artery disease in hypercholesterolemic pigs.     

Time course of changes in collateral blood flow and isolated vessel size and gene expression after femoral artery occlusion in rats

The objectives of this study were to assess the time course of enlargement and gene expression of a collateral vessel that enlarges following occlusion of the femoral artery and to relate these responses to the increases in collateral-dependent blood flow to the calf muscles in vivo. We employed exercise training to stimulate collateral vessel development. Rats were exercise trained or kept sedentary for various times of up to 25 days postbilateral occlusion (n=approximately 9/time point). Collateral blood flow to the calf muscles, determined with microspheres, increased modestly over the first few days to approximately 40 ml.min(-1).100 g(-1) in sedentary animals; the increase continued over time to approximately 80 ml.min(-1).100 g(-1) in the trained animals. Diameters of the isolated collateral vessels increased progressively over time, whereas an increased vessel compliance observed at low pressures was similar across time. These responses were greater in the trained animals. The time course of upregulation of vascular endothelial growth factor and placental growth factor, and particularly endothelial nitric oxide synthase and fms-like tyrosine kinase 1, mRNAs in the isolated collateral vessel implicates these factors as integral to the arteriogenic process. Collateral vessel enlargement and increased compliance at low pressures contribute to the enlarged circuit available for collateral blood flow. However, modulation of the functioning collateral vessel diameter, by smooth muscle tone, must occur to account for the observed increases in collateral blood flow measured in vivo.     

MAP-Kinase Activated Protein Kinase 2 Links Endothelial Activation and Monocyte/macrophage Recruitment in Arteriogenesis

Arteriogenesis, the growth of natural bypass arteries, is triggered by hemodynamic forces within vessels and requires a balanced inflammatory response, involving induction of the chemokine MCP-1 and recruitment of leukocytes. However, little is known how these processes are coordinated. The MAP-kinase-activated-proteinkinase-2 (MK2) is a critical regulator of inflammatory processes and might represent an important link between cytokine production and cell recruitment during postnatal arteriogenesis. Therefore, the present study investigated the functional role of MK2 during postnatal arteriogenesis. In a mouse model of hindlimb ischemia (HLI) MK2-deficiency (MK2KO) significantly impaired ischemic blood flow recovery and growth of collateral arteries as well as perivascular recruitment of mononuclear cells and macrophages. This was accompanied by induction of endothelial MCP-1 expression in wildtype (WT) but not in MK2KO collateral arteries. Following HLI, MK2 activation rapidly occured in the endothelium of growing WT arteries in vivo. In vitro, inflammatory cytokines and cyclic stretch activated MK2 in endothelial cells, which was required for stretch- and cytokine-induced release of MCP-1. In addition, a monocyte cell autonomous function of MK2 was uncovered potentially regulating MCP-1-dependent monocyte recruitment to vessels: MCP-1 stimulation of WT monocytes induced MK2 activation and monocyte migration in vitro. The latter was reduced in MK2KO monocytes, while in vivo MK2 was activated in monocytes recruited to collateral arteries. In conclusion, MK2 regulates postnatal arteriogenesis by controlling vascular recruitment of monocytes/macrophages in a dual manner: regulation of endothelial MCP-1 expression in response to hemodynamic and inflammatory forces as well as MCP-1 dependent monocyte migration.     

Resveratrol downregulates acute pulmonary thromboembolism-induced pulmonary artery hypertension via p38 mitogen-activated protein kinase and monocyte chemoattractant protein-1 signaling in rats

AimsIn the present study, we explored the hypothesis that initiation of PH involves the upregulation of monocyte chemoattractant protein-1 (MCP-1) in acute PTE. We evaluated the effects of resveratrol and the role of p38 mitogen-activated protein kinase (MAPK) in this process.Main methodsA rat model of acute PTE was established by infusion of an autologous blood clot into the pulmonary artery through a polyethylene catheter. Rats were randomly divided into 1, 4, and 8 hour time groups. Resveratrol, C1142 (a rodent chimeric mAb that neutralizes rat MCP-1) or SB203580 (a p38MAPK specific inhibitor) was administered to the animals beginning 1 h prior to the start of the acute PTE protocol. At each time point, the mean pulmonary artery pressure (mPAP), mRNA and protein expressions of MCP-1 were measured. The phosphorylation of p38 MAPK (p-pMAPK) was also detected.Key findingsAcute PTE elicited significant increases in mean pulmonary artery pressure (mPAP), and up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1) and phosphorylation of p38 mitogen-activated protein kinase (p-p38 MAPK). Administration of C1142 markedly reduced mPAP. Furthermore, pre-treatment of rats with resveratrol significantly reduced mPAP and down-regulated the expression of MCP-1, which was associated with robustly suppressed acute PTE-induced p-p38MAPK expression.SignificanceThese findings suggested that MCP-1 was involved in the formation of acute PTE-induced PH, and resveratrol down-regulated the expression of MCP-1 by inhibiting acute PTE-induced p-p38MAPK activation, which contributed to the decrease in PH.     

Impact of MCP-1 and CCR-2 gene polymorphisms on coronary artery disease susceptibility

Coronary artery disease (CAD) was the second leading cause of death during the last 3 years in Taiwan. Smooth muscle cells, monocytes/macrophages, and endothelial cells produce monocyte chemoattractant protein-1 (MCP-1) within atherosclerotic plaques following binding to the chemokine receptor-2 (CCR-2). Previous studies have well-documented the association between MCP-1 expression and susceptibility to, or clinicopathological features, of CAD. This study investigated the relationships between MCP-1-2518A/G and CCR-2-V64I genetic polymorphisms and CAD in the Taiwanese population. A total of 608 subjects, including 392 non-CAD controls and 216 patients with CAD, were recruited and subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to evaluate the effects of these two polymorphic variants on CAD. Results indicated a significant association between MCP-1 -2548 gene polymorphism and susceptibility to CAD. GG genotypes (OR = 1.629; 95 % CI = 1.003-2.644), or individuals with at least one G allele (OR = 1.511; 95 % CI = 1.006-2.270), had a higher risk of CAD as compared with AA genotypes. Results also revealed that subjects with at least one A allele of the V64I CCR2 gene polymorphism had significantly increased risk of CAD. G allele in MCP-1-2518 might contribute to higher prevalence of atrial fibrillation in CAD patients (OR = 4.254; p < 0.05). In conclusion, MCP-1-2518G and CCR-2 64I gene polymorphisms represent important factors in determining susceptibility to CAD, and the contribution of MCP-1-2518G could be through effects on atrial fibrillation in CAD patients.     

Curcumin attenuates oxidative stress following downhill running-induced muscle damage

Downhill running causes muscle damage, and induces oxidative stress and inflammatory reaction. Recently, it is shown that curcumin possesses anti-oxidant and anti-inflammatory potentials. Interestingly, curcumin reduces inflammatory cytokine concentrations in skeletal muscle after downhill running of mice. However, it is not known whether curcumin affects oxidative stress after downhill running-induced muscle damage. Therefore, the purpose of this study was to investigate the effects of curcumin on oxidative stress following downhill running induced-muscle damage. We also investigated whether curcumin affects macrophage infiltration via chemokines such as MCP-1 and CXCL14. Male C57BL/6 mice were divided into four groups; rest, rest plus curcumin, downhill running, or downhill running plus curcumin. Downhill running mice ran at 22 m/min, −15% grade on the treadmill for 150 min. Curcumin (3 mg) was administered in oral administration immediately after downhill running. Hydrogen peroxide concentration and NADPH-oxidase mRNA expression in the downhill running mice were significantly higher than those in the rest mice, but these variables were significantly attenuated by curcumin administration in downhill running mice. In addition, mRNA expression levels of MCP-1, CXCL14 and F4/80 reflecting presence of macrophages in the downhill running mice were significantly higher than those in the rest mice. However, MCP-1 and F4/80 mRNA expression levels were significantly attenuated by curcumin administration in downhill running mice. Curcumin may attenuate oxidative stress following downhill running-induced muscle damage.     

bFGF increases collateral blood flow in aged rats with femoral artery ligation

We tested the hypothesis that aged animals are as responsive as the young adult animals in expanding collateral vasculature under a similar treatment of basic fibroblast growth factor (bFGF). Two age groups of male Fischer 344 rats (11 mo old; n = 32, 23 mo old; n = 43) weighing approximately 385 g were subdivided into normal, acute ligation [femoral artery (FA) ligated 3 days before blood flow (BF) measurement] or ligated groups for 16 days and received recombinant human bFGF intra-arterial infusion at doses of 0, 0.5, 5, and 50 microg x kg(-1) x day(-1). BF was determined with (85)Sr- and (141)Ce-labeled microspheres during treadmill running at 15 and 20 m/min at 15% grade. Blood pressure (BP) values were approximately 149 and approximately 163 mmHg (p < 0.05); heart rates were approximately 496 and approximately 512 beats/min in the aged and young adult groups during running, respectively. Maximal collateral BF values were confirmed by no additional BF increase in the calf muscle at the higher speed. Ligation of the FA for 3 days reduced the BF reserve to the calf muscle by approximately 90%. Calf muscle BF was modestly greater (10 ml x min(-1) x 100 g(-1)) by 16 days in the carrier group. bFGF infusion expanded collateral BF in a dose-dependent manner with an increase of 33 and 42 ml x min(-1) x 100 g(-1) (P < 0.001) in the 5 and 50 microg x kg(-1) x day(-1) bFGF groups, respectively. Aged animals showed similar BF improvements as observed with the adult groups in response to ligation surgery and bFGF treatment. Our data indicate that the aged rats (approximately 23 mo old) remain responsive to exogenous bFGF induced in developing collateral-dependent BF as the young adult (approximately 11 mo old) controls. This suggests that the influence of bFGF in expanding collateral BF should not be preempted in the aged group, the population most affected by peripheral arterial insufficiency.     

Reactive oxygen species and ERK 1/2 mediate monocyte chemotactic protein-1-stimulated smooth muscle cell migration

Summary Monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for monocytes, is thought to play a major role in atherosclerosis, but whether its atherogenic effects involve the direct modulation of vascular smooth muscle cell (SMC) functions remains unclear. This study examined the effects of MCP-1 on the migration of cultured A7r5 SMCs and the signaling pathways involved. Addition of recombinant MCP-1 stimulated SMC migration in modified Boyden chambers coated with type I collagen in a concentration-dependent manner, with 10^–9 M being maximally effective. Using untreated A7r5 cells, two MCP-1 receptors, CCR2 and CCR4, were detected and MCP-1 secretion was significantly increased by stimulation with platelet-derived growth factor. MCP-1-stimulated A7r5 migration was completely blocked by the NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI), and dose-dependently inhibited by polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), suggesting a role for reactive oxygen species (ROS) in this process. During MCP-1 stimulation, ROS production increased rapidly, then gradually decayed over 60 min, and this effect was markedly decreased by pretreatment with DPI or PEG-SOD. Interestingly, U0126 and PD98059, which inhibit activation of extracellular signal-regulated kinases 1/2 (ERK 1/2), significantly inhibited MCP-1-activated ROS generation. Furthermore, transfection of an active mutant of MEK1 (ERK 1/2 kinase) markedly increased superoxide production in rat aortic smooth muscle cells, as detected by dihydroethydium staining, suggesting that ERK 1/2 activation stimulates ROS generation. ERK 1/2 activation was increased for at least 30 min in cells incubated with MCP-1, and this effect was abolished by U0126 or DPI pretreatment. These results demonstrate that MCP-1 is a chemoattractant for SMCs and that MCP-1-stimulated migration requires both ROS production and ERK 1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1.These authors contributed equally to this work.     

Identification of differentially expressed genes like cofilin2 in growing collateral arteries

Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbits by occlusion of the femoral artery. In order to analyze the differential gene expression in arteriogenesis, cDNA of collateral arteries 24h after femoral occlusion or sham operation was subjected to suppression subtractive hybridization (SSH). We demonstrated an upregulation of the U6 snRNA binding protein Lsm5, cytochrome b, an expressed sequence tag, and the actin-depolymerizing factor cofilin2 mRNA in collateral arteries 24h after femoral ligation. For cofilin2, we also detected an increase in the protein level and a localization predominantly in smooth muscle cells of collaterals. Simultaneously with the upregulation of cofilin2 we found a downregulation of the alpha-smooth muscle actin mRNA in growing collateral arteries. In summary, our data showed an augmented expression level of genes contributing to different fundamental processes of arteriogenesis.