The objective of this study was to investigate the effects of chronic inhibition of nitric oxide synthase (NOS) on cyclooxygenase-2 (COX-2) expression in the macula densa (MD) of swine, as well as the effects on expression of related proteins. Adult female Yucatan swine were given either tap water (control, n = 6) or water with NG-nitro-l-arginine methyl ester (L-NAME, 100 mg/liter, n = 5) for a minimum of 30 days. Duplicate samples of kidney were fixed or snap frozen. There was a significant ( P = .0082) upregulation of COX-2 mRNA expression in the MD of L-NAME, as well as an apparent increase in COX-2 protein. Plasma renin activity also increased with L-NAME treatment (control, 0.34 ± 0.08 ng/ml; L-NAME, 1.26 ± 0.03 ng/ml; P = .00000003). There were no differences between groups in expression of either inducible NOS or renin protein or in serum electrolyte concentrations. In conclusion, with chronic inhibition of NOS, COX-2 in MD is upregulated, perhaps to compensate for loss of nitric oxide. Increases in COX-2 products may counteract renal arteriolar constriction and sustain renin release.

Survivin, an inhibitor of apoptosis, is overexpressed in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Survivin expression in canine TCC has not been defined. This study was designed to compare survivin expression between canine TCC and normal urinary bladder tissue. Reverse-transcriptase polymerase chain reaction (PCR) and immunohistochemistry (IHC) were performed on fresh-frozen and formalin-fixed tissues, respectively. All TCC tissues (n = 6) and 11/22 normal tissues assessed by PCR were positive for survivin. This difference was not significant (P = 0.06). With regard to IHC, 28/41 TCC samples were positive for nuclear survivin, whereas 0/46 normal tissues had nuclear immunoreactivity (P < 0.001). Cytoplasmic immunoreactivity did not significantly differ between TCC (7/41) and normal tissues (17/46) (P = 0.07). We conclude that nuclear survivin is present in canine TCC, but not in normal bladder urothelium. Future studies will evaluate the role of nuclear survivin in TCC development and as a potential therapeutic target.

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by nitric oxide (NO) released by endothelial cell nitric oxide synthase (eNOS), and NO released by eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolic and cardiovascular systems of pigs and humans. To gain a better understanding of cardiovascular regulation by eNOS we produced pigs carrying an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag. Nuclear transfer was conducted to create these animals and the effects of two different oocyte activation treatments and two different culture systems were examined. Donor cells were electrically fused to the recipient oocytes. Electrical fusion/activation (1 mM calcium in mannitol: Treatment 1) and electrical fusion (0.1 mM calcium in mannitol)/chemical activation (200 µM Thimerosal for 10 min followed by 8 mM DTT for 30 min: Treatment 2) were used. Embryos were surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. Two cloned transgenic piglets were born from Treatment 1 and low oxygen, and another two from Treatment 2 and normal oxygen. PCR, RT-PCR, Western blotting and immunohistochemistry confirmed that the pigs were transgenic, made message, made the fusion protein and that the fusion protein localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. Thus both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system.

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.

Following the cessation of ovarian steroid hormone production meningeal microvascular networks of ovariectomized pigs undergo dramatic remodeling characterized by a 3-fold decrease in microvessel density, an increase in average microvessel size, and greater than 4-fold increase in vascular permeability. To elucidate the molecular mechanisms underpinning ovarian hormone dependent microvascular remodeling, we analyzed changes in expression of the PDGF/VEGF family growth factors and their receptors on systemic (serum) and local (tissue, large vessels, and microvessels) levels. Our results demonstrate differential patterns of regulation of PDGF, VEGF, and their receptors indicating that both systemic and local alterations in the expression of PDGF AB and BB and PDGF receptor alpha (αPDGFR) play key roles in regulating ovarian hormone dependent microvascular remodeling. In contrast, systemic and local changes in VEGF and VEGFR2/flk-1 appear to be minute and somewhat secondary to the changes in the PDGF/αPDGFR system. How autocrine or paracrine regulation of ER-interacting proteins generate cell-type and tissue-specific effects of estrogen remains to be determined. Supported by: AHA Heartland Affiliate Postdoctoral Fellowship, NASA NNJ05HF376, and NIH1C06RR017353-01

We previously demonstrated functional upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) with the development of early coronary atherosclerosis in vivo and as a prerequisite for platelet-derived growth factor BB (PDGF-BB) induced porcine coronary smooth muscle cell (CSMC) dedifferentiation and migration in vitro. Our objective was to examine the temporal regulation of IKCa1 and smooth muscle marker gene expression in porcine CSMC exposed to coronary balloon injury (CBI) in vivo or PDGF-BB in vitro. Two hours following CBI, laser capture microdissection of media from injured vessels demonstrated higher IKCa1, c-jun, and c-fos, but unaltered amounts of SMMHC mRNA compared to control media. PDGF-BB increased enrichment of H4 acetylation and c-jun to the AP-1 binding site of the IKCa1 promoter ~4-fold, an effect blocked by TRAM-34, a specific IKCa1 channel blocker. PDGF-BB increased IKCa1 and decreased myocardin mRNA at 30 minutes, but did not decrease smooth muscle myosin heavy chain (SMMHC) or smoothelin expression until 10 hours. In conclusion, these data indicate that AP-1 dependent upregulation of IKCa1 is an immediate and requisite factor initiating smooth muscle specific gene suppression, i.e. CSMC phenotypic modulation, during coronary injury. Supported by: NIH HL52490

ABSTRACTObjective: Exercise training has been shown to increase regional blood flow capacity to muscle tissue containing fibers that experience increased activity during exercise. The purpose of this study was to test the hypothesis that the increased blood flow capacity is partially the result of increases in arteriolar density (number of arterioles/mm2 of tissue), specifically in skeletal muscle tissue, with the largest relative increase in muscle fiber activity during training bouts.Methods: This hypothesis was tested by comparing and contrasting the effects of endurance exercise training (ET) and interval sprint training (IST) on arteriolar density in soleus muscle (S) red (Gr) and white (Gw) portions of gastrocnemius muscle of male Sprague Dawley rats. ET rats completed 10 weeks of treadmill training 30 m/min, 15% grade, 60 min/day, 5 days/week, while IST rats completed 10 weeks of IST consisting of six 2.5‐min exercise bouts, with 4.5‐min rest between bouts (60 m/min, 15% incline), 5 days/week. The hypothesis would be supported if ET increased arteriolar density in S and Gr and if IST increased arteriolar density in Gw.Results: ET increased arteriolar density above values of sedentary rats (SED) in both the Gw (ET = 0.93 ± 0.19 arterioles/μ m2; SED = 0.44 ± 0.09 arterioles/μ m2) and Gr (ET = 0.97 ± 0.1 arterioles/μ m2; SED = 0.51 ± 0.06 arterioles/μ m2) muscles, but not in S (ET = 1.69 ± 0.45 arterioles/μ m2; SED = 1.51 ± 0.34 arterioles/μ m2) muscle. In contrast, IST did not alter arteriolar density in Gw or Gr muscle tissue. Although arterial wall thickness was greater in S (3.95 ± 0.40 μ m) and Gr (6.24 ± 0.59 μ m) than Gw (2.76 ± 0.18 μ m), neither ET or IST altered mean wall thickness in either muscle.Conclusion: Increases in blood flow capacity produced in Gr and Gw by ET appear to be due in part to increased arteriolar density. In contrast, increased arteriolar density does not contribute to increased blood flow capacity of Gw in IST rats.

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by endothelial nitric oxide synthase (eNOS). In addition, eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolism, and cardiovascular systems of pigs and humans. The main goal of this study was to produce pigs that carry an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag so that a better understanding of cardiovascular regulation by eNOS can be gained. Nuclear transfer was conducted to create these animals, and the effects of two different oocyte activation treatments and two different culture systems were examined. Mature oocytes were purchased from BoMed, Inc. (Maidson, WI, USA) and enucleated by micromanipulation. Donor cells were injected into the perivitelline space and electrically fused to the recipient oocyte. Treatment 1 consisted of electrical fusion/activation in 1 mM calcium in mannitol; Treatment 2 consisted of electrical fusion in 0.1 mM calcium in mannitol, followed by chemical activation in 200 �M thimerosal for 10 min followed by 8 mM DTT for 30 min. The fusion rate (71% vs. 82%) was lower (P < 0.0001) in Treatment 2 than in Treatment 1. For those oocytes that fused, the cleavage rates (5.9% vs. 0.4%) at 14-18 h were higher (P < 0.0001) for Treatment 2. Fused oocytes were cultured for 12-18 h in 5% O2, 5% CO2, 90% N2 (low oxygen) or 5% CO2 in air (normal oxygen), and were then surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. In February four cloned transgenic piglets were born (birth weights were 495, 595, 965, and 685 g). Two were from Treatment 1 and low oxygen, and the other two were from Treatment 2 and normal oxygen, and all are currently healthy. PCR, RT-PCR, and Western blotting analysis confirmed that the pigs were transgenic, made message, and made the fusion protein. Immunohistochemistry confirmed that the fusion protein localized in the endothelial cells of the placental vasculature from the conceptuses as did the endogenous eNOS. In addition to the transgenic males, four wild-type females were born (birth weights were 585 (this one died on Day 4) 445, 930, and 585 g). All four were from Treatment 2 and low oxygen, and three are healthy. In conclusion, viable piglets were produced from both Treatments 1 and 2, as well as in both high and low oxygen conditions. Thus, both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system. This work was funded by a grant from the NIH RR18276 and Food for the 21st Century.

CASE DESCRIPTION:A 13-year-old llama was examined because of lethargy, inappetence, and syncope.CLINICAL FINDINGS:Physical examination revealed muffled heart and lung sounds and peripheral edema. Clinicopathologic abnormalities included lymphopenia, hyperglycemia, prerenal azotemia, mild hyponatremia, mild hypoalbuminemia, and high gamma-glutamyltransferase and creatine kinase activities. On ultrasonography, the liver appeared hyperechoic and ascites and pleural effusion were seen. Echocardiography revealed severe dilatation of the right atrium, right ventricle, and pulmonary artery; severe tricuspid regurgitation; and high right ventricular systolic pressure consistent with right-sided heart failure secondary to pulmonary hypertension.TREATMENT AND OUTCOME:Treatment with furosemide was attempted, but because of failing health, the llama was euthanized 4 weeks later. Macronodular cirrhosis of the liver, glomerulonephritis, and intimal fibrosis and medial hypertrophy of muscular pulmonary arteries were seen on histologic examination of postmortem specimens.CLINICAL RELEVANCE:Findings in this case were similar to those reported for human patients with portopulmonary hypertension secondary to hepatic cirrhosis. Pulmonary hypertension secondary to hepatic disease should be considered in the differential diagnosis of right-sided heart failure.

Glucocorticoids (GC) exert diverse cellular effects in response to both acute and chronic stress, the functional consequences of which have been implicated in the development of cardiovascular pathology such as hypertension and atherosclerosis. However, the mechanisms by which GCs activate divergent signaling pathways are poorly understood. The present study examined the direct effects of natural (cortisol) and synthetic (dexamethasone) GCs on protein kinase C (PKC) isoform expression in coronary arteries. Porcine right coronary arteries were treated in vitro for 18 h in the presence and absence of either dexamethasone (10, 100, or 500 nM) or cortisol (50, 125, 250, or 500 nM). PKC isoform levels and subcellular distribution were determined by immmunoblotting of whole cell homogenates and immunocytofluorescence using PKC-α, -βII, -ε, -δ, and -ζ specific antibodies. Dexamethasone caused a ∼4-fold increase in PKC-α, a ∼2.5-fold increase in PKC-βII, and a 2-fold increase in PKC-ε (p < 0.05). In contrast, dexamethasone had no effect on PKC-δ or PKC- ζ levels. Dexamethasone also caused an increase in the activity of PKC-α (285%), -βII (170%), and -ε (210%). Cortisol produced similar effects on PKC isoform expression. Confocal microscopy revealed that while dexamethasone altered localization patterns for PKC-α, -βII, and -ε, no such effect was observed for PKC-δ or PKC-ζ. The stimulatory effects of dexamethasone and cortisol on coronary PKC levels and translocation were prevented by the GC receptor (GR) blocker, RU486. These results demonstrate, for the first time, that GCs modulate coronary PKC expression and subcellular distribution in an isoform-specific manner through a GR-dependent mechanism.

We tested the hypothesis that exercise (Ex) training attenuates hypercholesterolemia-induced impairment of endothelium-dependent relaxation (EDR) in brachial (Br) arteries of adult male pigs by enhancing nitric oxide (NO)-mediated EDR. Adult male pigs were fed a normal-fat (NF) or high-fat/cholesterol (HF) diet for 20 wk. Four weeks after the diet was initiated, pigs were trained or remained sedentary (Sed) for 16 wk, yielding four groups: 1) NF-Sed, 2) NF-Ex, 3) HF-Sed, and 4) HF-Ex. EDR of Br artery rings was assessed in vitro with acetylcholine (ACh) and bradykinin (BK). ACh- and BK-induced relaxation was not impaired by HF; however, relaxation responses were enhanced by Ex in NF and HF arteries. To determine the mechanism(s) by which Ex improved EDR, ACh- and BK-induced relaxation was assessed in the presence of N-G-nitro-L-arginine methyl ester (L-NAME; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), or L-NAME + Indo. ACh- and BK-induced relaxation was inhibited by L-NAME, and L-NAME + Indo, in all groups of arteries. Indo did not inhibit ACh-induced relaxation in any group but did inhibit BK-induced relaxation in HF-Ex arteries. In the. presence Of L-NAME or L-NAME + Indo, ACh- and BK-induced relaxation in HF-Ex arteries remained greater than in HF-Sed arteries. However, in the presence of Indo, ACh-induced relaxation in HF-Ex arteries was no longer greater than in HF-Sed arteries. These results indicate that EDR is not impaired by hypercholesterolemia in Br arteries from adult male pigs; however, Ex improves EDR in HF Br arteries by enhancing production of endothelium-derived hyperpolarizing factor and/or prostacyclin.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the United States and is projected to become the leading cause of mortality in the world. Atherosclerosis is the most important single factor contributing to this disease burden. In this study, we characterize relationships between endothelial dysfunction and vascular disease in an animal model of diet-induced, early-stage atherosclerotic vascular disease. We tested the hypothesis that hypercholesterolaemia induces vascular disease and impairs endothelium-dependent relaxation (EDR) in conduit arteries of adult male Yucatan pigs. Pigs were fed a normal fat (NF) or high fat cholesterol (HFC) diet for 20-24 weeks. Results indicate that, while the HFC diet did not alter EDR in femoral or brachial arteries, EDR was significantly decreased in both carotid and coronary arteries. Sudanophilic fatty streaks were significantly present in the abdominal aorta and common carotid artery. Histopathology revealed increased intima-media thickness (IMT) and foam cell accumulation in Stary Stage I-III lesions in the abdominal aorta, common carotid artery and femoral arteries. In the coronary arteries, the accumulation of foam cells in Stary Stage I and II lesions resulted in a trend for increased IMT. There was no evidence of vascular disease in the brachial arteries. These results indicate that early stages of CVD (Stary Stage I-III) precede decreases in EDR induced by HFC diet, because femoral arteries exhibited foam cell accumulation and an increased IMT but no change in endothelial function.

Thyroid disease has profound effects on cardiovascular function. Hypo- and hyperthyroidism, for example, are associated with reduced and increased maximal endothelium-dependent vasodilation respectively. We therefore hypothesized that the capacity for vascular nitric oxide (NO) formation is decreased in hypothyroidism and increased in hyperthyroidism. To test this hypothesis, rats were made hypothyroid (HYPO) with propylthiouracil or hyperthyroid (HYPER) with triiodothyronine over 3-4 months. Compared with euthyroid control rats (EUT), HYPO exhibited blunted growth and lower citrate synthase activity in the soleus muscle; HYPER exhibited left ventricular hypertrophy and higher citrate synthase activity in the soleus muscle (P<0.05 for all effects). The capacity for NO formation was determined in aortic extracts by formation of [3H]L-citrulline from [3H]L-arginine, i.e. NO synthase (NOS) activity. Thyroid status modulated NOS activity (EUT, 36.8 +/- 5.5 fmol/h per mg protein; HYPO, 26.0 +/- 7.9; HYPER, 64.6 +/- 12.7; P<0.05, HYPER vs HYPO). Expression of endothelial and neural isoforms of NOS was modulated by thyroid status in a parallel fashion. Capacity for responding to NO was also determined via measuring cGMP concentration in aortae incubated with sodium nitroprusside. Stimulated cGMP formation was also modulated by thyroid status (EUT, 73.0 +/- 20.2 pmol/mg protein; HYPO, 152.4 +/- 48.7; HYPER, 10.4 +/- 2.6; P<0.05, HYPER vs HYPO). These data indicate that thyroid status alters capacities for both formation of and responding to NO. The former finding may contribute to previous findings concerning vascular function in thyroid disease states.

The two major theories of cancer metastasis, the seed and soil hypothesis and the mechanical trapping theory, view tumor cell adhesion to blood vessel endothelia and cancer cell aggregation as corresponding key components of the metastatic process. Here, we demonstrate in vitro, ex vivo, and in vivo that metastatic breast and prostate carcinoma cells form multicellular homotypic aggregates at the sites of their primary attachment to the endothelium. Our results suggest that metastatic cell heterotypic adhesion to the microvascular endothelium and homotypic aggregation represent two coordinated subsequent steps of the metastatic cascade mediated largely by similar molecular mechanisms, specifically by interactions of tumor-associated Thomsen-Friedenreich glycoantigen with the beta-galactoside-binding protein, galectin-3. In addition to inhibiting neoplastic cell adhesion to the endothelium and homotypic aggregation, disrupting this line of intercellular communication using synthetic Thomsen-Friedenreich antigen masking and Thomsen-Friedenreich antigen mimicking compounds greatly affects cancer cell clonogenic survival and growth as well. Thus, beta-galactoside-mediated intravascular heterotypic and homotypic tumor cell adhesive interactions at the sites of a primary attachment to the microvascular endothelium could play an important role during early stages of hematogenous cancer metastasis.

Because hypercholesterolemia can attenuate endothelial function and exercise training can augment endothelial function, we hypothesized that exercise training would improve endothelial function of coronary arterioles from pigs in the early stages of cardiovascular disease induced by a high-fat, high-cholesterol (HF) diet. Yucatan miniature swine were fed a normal-fat (NF) diet or HF diet (2% cholesterol) for 20 wk in which 8 and 46% of their calories were derived from fat, respectively. Both groups were subdivided into sedentary (Sed) or exercise-trained (Ex) groups. This resulted in four experimental groups: NFSed, NFEx, HFSed, and HFEx. Endothelial function was assessed in coronary arterioles 75-100 microm in diameter dissected from the left ventricular apex. Responses to endothelial-dependent dilation induced by bradykinin (BK), ADP, and flow were similar in all four groups, whereas dilation to aggregating platelets in the presence of indomethacin and ketanserin was attenuated in HFSed arterioles (P = 0.01). The attenuated response to aggregating platelets was prevented or reversed in HFEx arterioles (P = 0.03). In HFSed arterioles, BK induced release of an indomethacin-sensitive prostanoid constrictor. In contrast, after exercise training, there was no evidence of this constrictor and BK-induced release of an indomethacin-sensitive prostanoid dilator in HFEx arterioles (P = 0.04). Endothelial nitric oxide synthase protein in arterioles was significantly reduced in HF groups (P < 0.05) and increased in Ex groups (P < 0.05). Interestingly, the relative contribution of nitric oxide to BK-induced dilation, as assessed with nitro-L-arginine methyl ester, was similar in arterioles in the NF, HF, Sed, and Ex groups. These results suggest that, in the early stages of cardiovascular disease, a high-fat, high-cholesterol diet has modest effects on endothelial-dependent dilation in coronary arterioles; nonetheless, these effects are prevented or reversed with exercise training.