Fluid shear stress upregulates placental growth factor in the vessel wall via NADPH oxidase 4.

Placental growth factor (PLGF), a potent stimulator of arteriogenesis, is upregulated during outward arterial remodeling. Increased fluid shear stress (FSS) is a key physiological stimulus for arteriogenesis. However, the role of FSS in regulating PLGF expression is unknown. To test the hypothesis that FSS regulates PLGF expression in vascular cells and to identify the signaling pathways involved, human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells were cultured on either side of porous Transwell inserts. HCAEC were then exposed to pulsatile FSS of 0.07 Pa ("normal," mimicking flow through quiescent collaterals), 1.24 Pa ("high," mimicking increased flow in remodeling collaterals), or 0.00 Pa ("static") for 2 h. High FSS increased secreted PLGF protein ∼1.4-fold compared with static control (n = 5, P < 0.01), while normal FSS had no significant effect on PLGF. Similarly, high flow stimulated PLGF mRNA expression nearly twofold in isolated mouse mesenteric arterioles. PLGF knockdown using siRNA revealed that HCAEC were the primary source of PLGF in cocultures (n = 5, P < 0.01). Both H2O2 and nitric oxide production were increased by FSS compared with static control (n = 5, P < 0.05). N(G)-nitro-l-arginine methyl ester (100 µM) had no significant effect on the FSS-induced increase in PLGF. In contrast, both catalase (500 U/ml) and diphenyleneiodonium (5 µM) attenuated the effects of FSS on PLGF protein in cocultures. Diphenyleneiodonium also blocked the effect of high flow to upregulate PLGF mRNA in isolated arterioles. Further studies identified NADPH oxidase 4 as a source of reactive oxygen species for this pathway. We conclude that FSS regulates PLGF expression via NADPH oxidase 4 and reactive oxygen species signaling.

Placenta growth factor and vascular endothelial growth factor a have differential, cell-type specific patterns of expression in vascular cells.

OBJECTIVE: PLGF, a VEGF-A related protein, mediates collateral enlargement via monocytes but plays little role in capillary proliferation. In contrast, VEGF-A mediates both collateral enlargement and capillary proliferation. PLGF has been less thoroughly studied than VEGF-A, and questions remain regarding its regulation and function. Therefore, our goal was to characterize the expression of PLGF by vascular cells. We hypothesized that vascular SMC would express more PLGF than EC, since VEGF-A is primarily expressed by non-EC. METHODS: We compared PLGF and VEGF-A across eight EC and SMC lines, then knocked down PLGF and evaluated cell function. We also assessed the effect of hypoxia on PLGF expression and promoter activity. RESULTS: PLGF was most highly expressed in EC, whereas VEGF-A was most highly expressed in SMC. PLGF knockdown did not affect EC number, migration, or tube formation, but reduced monocyte migration toward EC. Monocyte migration was rescued by exogenous PLGF. Hypoxia increased PLGF protein without activating PLGF gene transcription. CONCLUSIONS: PLGF and VEGF-A have distinct patterns of expression in vascular cells. EC derived PLGF may function primarily in communication between EC and circulating cells. Hypoxia increases EC PLGF expression posttranscriptionally.

Post-transcriptional regulation of placenta growth factor mRNA by hydrogen peroxide.

In tissues containing pre-existing collateral vessels, occlusion of an upstream supply artery results in diversion of blood flow through these vessels, protecting the distal tissue from ischemia. The sudden rise in blood flow through collateral vessels exerts shear stress upon the vessel wall, thereby providing the initial stimulus for arteriogenesis. Arteriogenesis, the structural expansion of collateral circulation, involves smooth muscle cell (SMC) proliferation which leads to increased vessel diameter and wall thickness. Since shear is sensed at the level of endothelial cells (EC), communication from EC to the underlying SMC must occur as part of this process. We previously reported that endothelial cells (EC) exposed to shear stress release hydrogen peroxide (H(2)O(2)), and that H(2)O(2) can signal vascular SMC to increase gene and protein expression of placenta growth factor (PLGF), a known mediator of arteriogenesis. The purpose of the current study was to further elucidate the mechanism whereby PLGF is regulated by H(2)O(2). We found that a single, physiological dose of H(2)O(2) increases PLGF mRNA half-life, but has no effect on PLGF promoter activity, in human coronary artery SMC (CASMC). We further demonstrated that the H(2)O(2)-induced increase in PLGF mRNA levels partially relies on p38 MAPK, JNK and ERK1/2 pathways. Finally, we showed that chronic exposure to pathological levels of H(2)O(2) further increases PLGF mRNA levels, but does not result in a corresponding increase in PLGF secreted protein. These data suggest that PLGF regulation has an important translational component. To our knowledge, this is the first study to characterize post-transcriptional regulation of PLGF mRNA by H(2)O(2) in vascular SMC. These findings provide new insights into the regulation of this important growth factor and increase our understanding of PLGF-driven arteriogenesis.

Exercise training and peripheral arterial disease.

Peripheral arterial disease (PAD) is a common vascular disease that reduces blood flow capacity to the legs of patients. PAD leads to exercise intolerance that can progress in severity to greatly limit mobility, and in advanced cases leads to frank ischemia with pain at rest. It is estimated that 12 to 15 million people in the United States are diagnosed with PAD, with a much larger population that is undiagnosed. The presence of PAD predicts a 50% to 1500% increase in morbidity and mortality, depending on severity. Treatment of patients with PAD is limited to modification of cardiovascular disease risk factors, pharmacological intervention, surgery, and exercise therapy. Extended exercise programs that involve walking approximately five times per week, at a significant intensity that requires frequent rest periods, are most significant. Preclinical studies and virtually all clinical trials demonstrate the benefits of exercise therapy, including improved walking tolerance, modified inflammatory/hemostatic markers, enhanced vasoresponsiveness, adaptations within the limb (angiogenesis, arteriogenesis, and mitochondrial synthesis) that enhance oxygen delivery and metabolic responses, potentially delayed progression of the disease, enhanced quality of life indices, and extended longevity. A synthesis is provided as to how these adaptations can develop in the context of our current state of knowledge and events known to be orchestrated by exercise. The benefits are so compelling that exercise prescription should be an essential option presented to patients with PAD in the absence of contraindications. Obviously, selecting for a lifestyle pattern that includes enhanced physical activity prior to the advance of PAD limitations is the most desirable and beneficial.

Placenta growth factor expression is regulated by hydrogen peroxide in vascular smooth muscle cells.

When supply arteries become occluded, blood is diverted through preexisting collateral vessels. Shear stress arising from this increase in blood flow provides the initial physiological stimulus for expansion of the collateral circulation, a process termed arteriogenesis. Endothelial cells (EC) respond to increased shear stress by releasing a variety of mediators that can act on underlying smooth muscle cells (SMC). Placenta growth factor (PLGF) is known to mediate certain aspects of arteriogenesis, such as recruitment of monocytes to the vessel wall. Therefore, we tested whether SMC PLGF expression is influenced by mediators released by EC. We used A10 SMC cultured with medium that had been conditioned by EOMA EC for 4 days as a model. We found that EC-conditioned medium is able to upregulate PLGF gene expression in A10 SMC. Further experiments identified hydrogen peroxide (H(2)O(2)) as a key mediator of this response. We confirmed the physiological relevance of this mechanism in primary human coronary artery SMCs by demonstrating that exogenous H(2)O(2) specifically upregulates PLGF gene and protein expression. We also demonstrated that the physiological stimulus of shear stress raises endogenous H(2)O(2) levels in media into the range found to increase PLGF expression. In this study, we demonstrate that EC-released H(2)O(2) acts as a positive regulator of PLGF gene and protein expression in vascular SMC. To our knowledge, this is the first study to describe H(2)O(2) as a regulator of PLGF expression and therefore an upstream mediator of PLGF-driven arteriogenesis.

Leukemia inhibitory factor is upregulated in coronary arteries of Ossabaw miniature swine after stent placement.

Leukemia inhibitory factor (LIF), an IL-6 class cytokine, is reported to be antiatherosclerotic. Thus, we hypothesized that LIF expression might be altered during in-stent neointimal hyperplasia. Ossabaw miniature swine, a unique large-animal model of metabolic syndrome and cardiovascular disease, were used for these studies. Bare-metal stents were deployed in the left anterior descending and left circumflex coronary arteries. Stents were expanded to either 1.0 x luminal diameter (in accordance with current clinical practice) or 1.3 x (overexpansion). The development of in-stent neointimal hyperplasia was assessed 28-day postimplantation using intravascular ultrasound. The atherosclerotic coverage of the vessel wall was approximately five-fold higher in 1.0 x stents and approximately nine-fold higher in 1.3 x stents 4 weeks after deployment, compared with the same segments before stenting. LIF mRNA was elevated approximately 11-fold in stented segments, relative to unstented epicardial coronary arteries. LIF expression and the intima : media ratio were strongly correlated in 1.0 x stented vessels. Further studies to investigate the nature of the association between LIF and neointimal hyperplasia revealed that vascular smooth muscle cell proliferation was inhibited by LIF treatment in an in-vitro model of atherosclerosis (coronary artery organ culture). These novel and clinically relevant studies show that elevated LIF gene expression is predictive for in-stent neointimal hyperplasia, and suggest that LIF upregulation may be a compensatory mechanism in this setting.

Adenosine A1 receptors in neointimal hyperplasia and in-stent stenosis in Ossabaw miniature swine.

BACKGROUND: Stent-induced neointimal hyperplasia is a major cause of morbidity following stent deployment in patients with coronary artery disease. Importantly, however, mechanisms underlying stent-induced neointimal hyperplasia are unclear. This pathological response to stent placement is more aggressive when stents are over-expanded, suggesting that vascular injury may play a role. In this study we tested the hypothesis that adenosine A1 receptor upregulation is associated with neointimal hyperplasia within coronary artery stents. METHODS: Adult male Ossabaw swine were used as our experimental model. Neointima formation and gene expression were studied 4 weeks after coronary stents were placed at 1.0x or 1.3x luminal diameter. RESULTS: Neointima formation was observed in 1.0x stents and more than doubled in 1.3x stents, thus verifying the response to overexpansion injury. A1 receptor mRNA was increased four-fold and seven-fold in stents at 1.0x and 1.3x luminal diameter, suggesting that increased A1 receptor activity might contribute to stent-induced neointimal hyperplasia. Coronary artery organ culture model of arterial injury demonstrated A1 receptor activation increased DNA synthesis three-fold, an effect abolished by A1 receptor antagonism. CONCLUSION: Our data indicate that A1 receptor expression is increased within stents and that activation of A1 receptors increases smooth muscle cell proliferation. We suggest that inhibition of A1 receptor signaling may be a promising therapeutic target for management of in-stent stenosis.

Gender and genetic differences in bladder smooth muscle PPAR mRNA in a porcine model of the metabolic syndrome.

The metabolic syndrome and diabetes are associated with bladder dysfunction in many people. Peroxisome proliferator-activated receptors (PPARs) may play a role in the effects of the metabolic syndrome on bladder smooth muscle (BSM). The purpose of this study was to determine if there are gender and genetic differences in PPAR levels in BSM. We measured PPAR levels using quantitative PCR in BSM from male Yucatan swine and male and female Ossabaw Island swine, which is a model for the metabolic syndrome. Male Ossabaw swine had 0.732 +/- 0.111 the amount of PPAR-alpha mRNA as male Yucatan swine (P < 0.05), suggesting a genetic difference in PPAR-alpha levels. This difference may possibly contribute to the incidence of metabolic syndrome in the Ossabaw model compared to the Yucatan model. PPAR-delta mRNA was 2-fold higher in male Ossabaw swine than in female Ossabaw swine, with no significant differences in PPAR-alpha levels. However, PPAR-gamma mRNA was 4.067 +/- 0.134 times higher in female Ossabaw swine than in their male counterparts (P < 0.001). Changing the percentage of calories derived from fat did not alter any PPAR mRNA levels. Thus, PPAR-delta and PPAR-gamma mRNA levels in male and female Ossabaw swine BSM are not only different, but may also result in gender differences in lipid metabolism in bladder smooth muscle. We conclude that PPAR profiles in BSM may contribute to the susceptibility of BSM to lipotoxicity in the metabolic syndrome.

Overexpression of caveolin-1 results in increased plasma membrane targeting of glycolytic enzymes: the structural basis for a membrane associated metabolic compartment.

Although membrane-associated glycolysis has been observed in a variety of cell types, the mechanism of localization of glycolytic enzymes to the plasma membrane is not known. We hypothesized that caveolin-1 (CAV-1) serves as a scaffolding protein for glycolytic enzymes and may play a role in the organization of cell metabolism. To test this hypothesis, we over-expressed CAV-1 in cultured A7r5 (rat aorta vascular smooth muscle; VSM) cells. Confocal immunofluorescence microscopy was used to study the distribution of phosphofructokinase (PFK) and CAV-1 in the transfected cells. Areas of interest (AOI) were analyzed in a central Z-plane across the cell transversing the perinuclear region. To quantify any shift in PFK localization resulting from CAV-1 over-expression, we calculated a periphery to center (PC) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found the PC index to be 1.92 +/- 0.57 (mean +/- SEM, N = 8) for transfected cells and 0.59 +/- 0.05 (mean +/- SEM, N = 11) for control cells. Colocalization analysis demonstrated that the percentage of PFK associated with CAV-1 increased in transfected cells compared to control cells. The localization of aldolase (ALD) was also shifted towards the plasma membrane (and colocalized with PFK) in CAV-1 over-expressing cells. These results demonstrate that CAV-1 creates binding sites for PFK and ALD that may be of higher affinity than those binding sites localized in the cytoplasm. We conclude that CAV-1 functions as a scaffolding protein for PFK, ALD and perhaps other glycolytic enzymes, either through direct interaction or accessory proteins, thus contributing to compartmented metabolism in vascular smooth muscle.

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.

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.

Exercise-induced vascular remodeling.

Exercise produces a powerful angiogenic stimulus within the active muscle that leads to a functionally important increase in capillarity. Further, exercise can increase flow capacity by enlarging the caliber of arterial supply vessels. These adaptations are achieved by the processes of angiogenesis and arteriogenesis, respectively.

Angiogenic growth factor expression in rat skeletal muscle in response to exercise training.

Angiogenesis occurs in skeletal muscle in response to exercise training. To gain insight into the regulation of this process, we evaluated the mRNA expression of factors implicated in angiogenesis over the course of a training program. We studied sedentary control (n = 17) rats and both sedentary (n = 18) and exercise-trained (n = 48) rats with bilateral femoral artery ligation. Training consisted of treadmill exercise (4 times/day, 1-24 days). Basal mRNA expression in sedentary control muscle was inversely related to muscle vascularity. Angiogenesis was histologically evident in trained white gastrocnemius muscle by day 12. Training produced initial three- to sixfold increases in VEGF, VEGF receptors (KDR and Flt), the angiopoietin receptor (Tie-2), and endothelial nitric oxide synthase mRNA, which dissipated before the increase in capillarity, and a substantial (30- to 50-fold) but transient upregulation of monocyte chemoattractant protein 1 mRNA. These results emphasize the importance of early events in regulating angiogenesis. However, we observed a sustained elevation of the angiopoietin 2-to-angiopoietin 1 ratio, suggesting continued vascular destabilization. The response to exercise was (in general) tempered in high-oxidative muscles. These findings place importance on cellular events coupled to the onset of angiogenesis.