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Biomedical subjects

Duncan J Stewart

Publications and source records attributed to Duncan J Stewart.

At least 19 recordsLinked to original sources
PubMed2007

Increased cerebral and renal endothelial nitric oxide synthase gene expression after cardiopulmonary bypass in the rat.

OBJECTIVE: Hemodilution and endothelial nitric oxide synthase genetic polymorphism may contribute to cerebral and renal injury after cardiopulmonary bypass. This study tested the hypothesis that cardiopulmonary bypass and anemia stimulate an increase in cerebral and renal endothelial nitric oxide synthase gene expression in an experimental model of cardiopulmonary bypass. METHODS: Anesthetized rats underwent a sham procedure without cardiopulmonary bypass (sham, n = 5), normothermic bypass for 1 hour (CPB, n = 7), or bypass plus hemodilutional anemia (CPB anemia, n = 9). After 24 hours of recovery, RNA was extracted from the cerebral cortex, renal cortex, and renal medulla. Quantitative reverse transcriptase polymerase chain reaction was used to assess endothelial nitric oxide synthase messenger RNA levels in brain and kidney tissues. RESULTS: The hemoglobin concentration of anemic CPB rats was significantly lower than that of nonanemic rats on bypass (64 +/- 5 vs 99 +/- 8 g x L(-1), P < .001). Cerebral cortical endothelial nitric oxide synthase messenger RNA levels were increased after cardiopulmonary bypass relative to those of the sham group (11.2 +/- 4.2 vs 6.3 +/- 1.5 fg, P = .031), without a further increase in anemic rats. Renal medullary endothelial nitric oxide synthase messenger RNA levels were significantly higher in the CPB anemia group than in the sham and CPB groups (7.1 +/- 4.4 fg vs 1.8 +/- 0.4 fg vs 3.0 +/- 0.6 fg, P < .001). Renal cortical endothelial nitric oxide synthase messenger RNA levels did not change significantly. CONCLUSIONS: Normothermic cardiopulmonary bypass was associated with higher endothelial nitric oxide synthase messenger RNA levels in kidney and brain than was the sham procedure 24 hours after cardiopulmonary bypass. Anemia accentuated the increase in renal medullary, but not cerebral cortical, endothelial nitric oxide synthase expression. These data provide an approach for exploring potential mechanisms by which endothelial nitric oxide synthase may contribute to renal and cerebral dysfunction after cardiopulmonary bypass and anemia.

Animals↗
PubMed2006

Adiponectin and cardiovascular disease: state of the art?

The cardiometabolic syndrome, associated with increased cardiovascular disease risk in the industrialized world, is estimated to affect one in four adults. Although the mechanisms linking obesity and cardiovascular disease remain unclear, research continues to unravel the molecular pathways behind this pandemic. Adipose tissue has emerged as a metabolically active participant in mediating vascular complications, serving as an active endocrine and paracrine organ secreting adipokines, which participate in diverse metabolic processes. Among these adipokines is adiponectin, which seems to possess antiatherogenic and anti-inflammatory effects and may be protective against cardiovascular disease development. The current review describes the pathophysiology of adiponectin in atherosclerotic disease.

Adiponectin↗
PubMed2006

Increased expression of HIF-1alpha, nNOS, and VEGF in the cerebral cortex of anemic rats.

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial Pa(O(2)) values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 +/- 2 g/l. In anemic rats, cerebral cortical HIF-1alpha protein levels were increased, relative to controls (1.7 +/- 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 +/- 0.2- and 1.5 +/- 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1alpha and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 +/- 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1alpha protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.

Anemia↗
PubMed2006

Microvascular regeneration in established pulmonary hypertension by angiogenic gene transfer.

Pulmonary arterial hypertension (PAH) is characterized by widespread loss of pulmonary microvasculature. Therefore we hypothesized that angiogenic gene therapy would reverse established PAH, in part restoring the lung microcirculation. Three weeks after monocrotaline (MCT) treatment, Fisher 344 rats were randomized to receive a total of either 1.5 x 10(6) syngeneic fibroblasts (FB) transfected with vascular endothelial growth factor A (VEGF), endothelial NO synthase (eNOS), or null-plasmid transfected FBs. Right ventricular systolic pressure (RVSP) was similarly increased in all MCT-treated groups at the time of gene transfer. Animals receiving the null-vector progressed to severe PAH by Day 35 (P < 0.001). In contrast, eNOS gene transfer significantly reduced RVSP at Day 35 compared with Day 21, whereas VEGF prevented further increases in RVSP over the subsequent 2 wk but did not reverse established PAH. RV hypertrophy was significantly reduced in both the eNOS-treated and VEGF-treated groups compared with the null-transfected controls. Fluorescent microangiography revealed widespread occlusion of the pre-capillary arterioles 21 d after MCT treatment, and animals receiving eNOS gene transfer exhibited the greatest improvement in the arteriolar architecture and capillary perfusion at Day 35. Cell-based eNOS gene transfer was more effective than VEGF in reversing established PAH, associated with evidence of regeneration of pulmonary microcirculation.

Animals↗
PubMed2006

Adventitial microvessel formation after coronary stenting and the effects of SU11218, a tyrosine kinase inhibitor.

OBJECTIVES: The aim of this study was to delineate the temporal profile of adventitial microvessel (Ad-MV) formation after stenting, its relationship to arterial wall hypoxia, and the effects of a tyrosine kinase inhibitor (TKI), SU11218, on Ad-MV and in-stent intimal hyperplasia (IH). BACKGROUND: Adventitial microvessels have been reported after arterial injury; however, the underlying stimulus for this response and its relationship to IH is unknown. METHODS: Coronary stenting was performed in 40 pigs randomized to SU11218 (n = 20) or placebo (n = 20). Vessel wall hypoxia was assessed by pimonidazole adducts and hypoxia-inducible factor (HIF)-1 alpha expression. Adventitial microvessels were quantified by three-dimensional microscopic computed tomography (3D micro CT). Intimal hyperplasia was measured by intravascular ultrasound (IVUS), 3D micro CT, and morphometry. The effects of SU11218 were assessed in vitro on smooth muscle cell (SMC) and endothelial cell (EC) functions and in vivo on Ad-MV and IH. RESULTS: Hypoxia was evident in the vessel wall at 48 h and persisted for four weeks. Adventitial microvessels increased significantly at one week (24 +/- 7 microvessels/segment) and four weeks (23 +/- 7 microvessels/segment) compared with uninjured arteries (16 +/- 2 microvessels/segment; p < 0.001) and correlated with IH (r = 0.77, p < 0.001). The TKI SU11218 inhibited platelet-derived growth factor receptor-beta phosphorylation, EC and SMC DNA synthesis, and migration in a dose-dependent manner in vitro and significantly inhibited Ad-MV (16 +/- 5 vs. 23 +/- 7 microvessels/segment in placebo, p < 0.001) and produced approximately 80% reduction in IH (0.52 +/- 0.51 mm2 vs. 2.47 +/- 1.66 mm2 in placebo, p < 0.001) at four weeks in vivo. CONCLUSIONS: Arterial stenting causes arterial wall hypoxia followed by Ad-MV formation. The TKI SU11218 inhibits both Ad-MV formation and IH and represents a promising therapeutic agent to prevent in-stent restenosis.

Animals↗
PubMed2006

Defective lung vascular development in endothelial nitric oxide synthase-deficient mice.

Normal blood vessel formation is required for the development of nearly all organs during embryogenesis, including lung. Neonatal diseases such as persistent pulmonary hypertension of the newborn and alveolar capillary dysplasia are thought to result in large part from a failure of normal lung vascular development. Therefore, in the past decade, there has been increasing interest in studying the mechanisms underlying the development of the lung circulation to better understand the pathogenesis of these often lethal neonatal lung diseases. It has now been well accepted that in addition to its protean roles in the maintenance of vascular homeostasis, endothelium-derived nitric oxide also plays a pivotal role in postnatal angiogenesis, mediating downstream signaling in response to classic angiogenic factors. Recent findings in endothelial nitric oxide synthase (eNOS)-deficient mice point to a novel and previously unrecognized role of eNOS-NO pathway in fetal lung vascular development and lung morphogenesis. The lung phenotype of eNOS mutants closely resembles alveolar capillary dysplasia in humans, a universally fatal form of persistent pulmonary hypertension of the newborn that presents with defective lung vascular development and respiratory distress in newborn. We anticipate that these new insights into the basic mechanisms of lung vascular development may lead to novel therapeutic strategies for neonatal lung diseases.

Animals↗
PubMed2006

Fluorescent microangiography (FMA): an improved tool to visualize the pulmonary microvasculature.

Visualization of the complex lung microvasculature and resolution of its three-dimensional architecture remains a difficult experimental challenge. We present a novel fluorescent microscopy technique to visualize both the normal and diseased pulmonary microvasculature. Physiologically relevant pulmonary perfusion conditions were applied using a low-viscosity perfusate infused under continuous airway ventilation. Intensely fluorescent polystyrene microspheres, confined to the vascular space, were imaged through confocal optical sectioning of 200 microm-thick lung sections. We applied this technique to rat lungs and the markedly enhanced depth of field in projected images allowed us to follow vascular branching patterns in both normal lungs and lungs from animals with experimentally induced pulmonary arterial hypertension. In addition, this method allowed complementary immunostaining and identification of cellular components surrounding the blood vessels. Fluorescent microangiography is a widely applicable and quantitative tool for the study of vascular changes in animal models of pulmonary disease.

Animals↗
PubMed2006

Positive-contrast imaging in the rabbit hind-limb of transplanted cells bearing endocytosed superparamagnetic beads.

Positive-contrast methods can augment negative-contrast methods for CMR evaluation of transplanted cell fate in vivo. This study correlated positive-contrast signal characteristics in the rabbit hind-limb with xenograft cell number (rat fibroblasts bearing endocytosed 1 microm Bang's particles) at different stages of Gd-DTPA administration and 1 mm2 in-plane resolution (2 minute scans, 1.5 Tesla). Linear regressions modeled satisfactorily the cell number dependencies of signal-to-noise (SNR) and area-of-enhancement (R > 0.83), over approximately a 50-fold fluctuation in cell number to a lowest detected limit of 25k cells during Gd-DTPA administration. Gd-DTPA administration elevated the slopes of parameter regressions by four or more standard deviations.

Animals↗
PubMed2006

Growth factor-induced therapeutic neovascularization for ischaemic vascular disease: time for a re-evaluation?

PURPOSE OF REVIEW: Therapeutic angiogenesis and arteriogenesis represent an alternative treatment modality for patients with advanced ischaemic coronary or peripheral artery occlusive disease, who are unsuitable for standard revascularization procedures. RECENT DEVELOPMENTS: Proof-of-concept evidence for therapeutic growth factor, both gene and protein-mediated neovascularization was provided in animal models of chronic myocardial and hindlimb ischaemia. Early human, phase I, trials utilizing the prototypical growth factor families, vascular endothelial growth factor and fibroblast growth factor, documented safety and suggested improvements in anginal symptoms and functional status. Large, randomized, placebo-controlled phase II/III clinical trials have, however, yielded variable results as such studies have suffered from significant limitations in therapeutic approach or design, which limits the ability to draw firm conclusions. SUMMARY: Future trials must incorporate robust delivery strategies and address issues of study design including proper patient selection. Laboratory-based refinements in therapy, including a focus on the promotion of arteriogenesis and the modification of patient 'endotheliopathy', will all further enhance the potential of therapeutic neovascularization strategies.

Angiogenesis Inducing Agents↗
PubMed2006

Regulation of endothelin-1 by angiopoietin-1: implications for inflammation.

Endothelin-1 (ET-1) is increasingly recognized as a proinflammatory mediator in various diseases, such as atherosclerosis and acute respiratory distress syndrome (ARDS). Angiopoietin-1 (Ang-1), a ligand of the endothelial receptor Tie2, inhibits endothelial apoptosis, reduces vascular leakage, and suppresses the induction of inflammatory markers, indicating that it has diverse vasoprotective, anti-inflammatory actions. Thus, we examined the effects of Ang-1 on ET-1 production in vitro and in vivo and investigated cell-based gene transfer of Ang-1 in a rat model of lipopolysaccharide (LPS)-induced ARDS. Cultured human endothelial cells were treated with recombinant Ang-1 with or without tumor necrosis factor-alpha (TNF-alpha) (100 U/ml). ET-1 release into the culture medium after 24 hrs was determined by enzyme-linked immunosorbent assay. Levels of preproendothelin-1 (ppET-1) mRNA were measured by quantitative reverse transcription-polymerase chain reaction. Fisher344 rats were subjected to cell-based gene transfer to the lung circulation by injecting syngeneic fibroblasts transfected with Ang-1 cDNA or a null plasmid vector. After 24 hrs, LPS (100 microg/kg body wt) was instilled intratracheally to induce pulmonary inflammation. Bronchoalveolar lavage was performed 6 hrs later, and lungs were harvested for histologic and molecular analyses. ET-1 release from cultured endothelial cells was dose-dependently reduced by Ang-1, which also prevented induction of ET-1 release by TNF-alpha (P < 0.05). RNA expression of ppET-1 was similarly reduced. In LPS-challenged lungs, ppET-1 RNA was induced 3.4-fold, and ET-1 protein in lavage fluid was increased 5.6-fold (P < 0.05). Ang-1 gene transfer attenuated the LPS-induced increases in ppET-1 RNA and lavage ET-1 protein by 34% and 33%, respectively (P < 0.05). The downregulation of ET-1 correlated with the amelioration of pulmonary inflammation, as indicated by reductions in leukocyte infiltration (by 43%) and intra-alveolar septal thickening (by 40%). These results show that ET-1 transcript and protein levels are downregulated by Ang-1 in both in vitro and in vivo systems and that cell-based Ang-1 gene transfer markedly ameliorated inflammation in vivo in an experimental model of ARDS. Thus, cell-based gene transfer of Ang-1 may provide a novel treatment strategy for ARDS by attenuating vascular inflammation via suppression of ET-1.

Angiopoietin-1↗
PubMed2005

Bone morphogenetic protein receptor-2 signaling promotes pulmonary arterial endothelial cell survival: implications for loss-of-function mutations in the pathogenesis of pulmonary hypertension.

Mutations in the bone morphogenetic protein (BMP) receptor-2 (BMPR2) have been found in patients with idiopathic pulmonary arterial hypertension (IPAH); however, the mechanistic link between loss of BMPR2 signaling and the development of pulmonary arterial hypertension is unclear. We hypothesized that, contrary to smooth muscle cells, this pathway promotes survival in pulmonary artery endothelial cells (ECs) and loss of BMPR2 signaling will predispose to EC apoptosis. ECs were treated with BMP-2 or BMP-7 (200 ng/mL) for 24 hours in regular or serum-free (SF) medium, with and without addition of tumor necrosis factor alpha, and apoptosis was assessed by flow cytometry (Annexin V), TUNEL, or caspase-3 activity. Treatment for 24 hours in SF medium increased apoptosis, and both BMP-2 and BMP-7 significantly reduced apoptosis in response to serum deprivation to levels not different from serum controls. Transfection with 5 microg of small interfering RNAs for BMPR2 produced specific gene silencing assessed by RT-PCR and Western blot analysis. BMPR2 gene silencing increased apoptosis almost 3-fold (P=0.0027), even in the presence of serum. Circulating endothelial progenitor cells (EPCs) isolated from normal subjects or patients with IPAH were differentiated in culture for 7 days and apoptosis was determined in the presence and absence of BMPs. BMP-2 reduced apoptosis induced by serum withdrawal in EPCs from normal subjects but not in EPCs isolated from patients with IPAH. These results support the hypothesis that loss-of-function mutations in BMPR2 could lead to increased pulmonary EC apoptosis, representing a possible initiating mechanism in the pathogenesis of pulmonary arterial hypertension.

Adult↗
PubMed2005

Phospho-akt expression is associated with a favorable outcome in non-small cell lung cancer.

Akt, a Serine/Threonine protein kinase, mediates growth factor-associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non-small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables. Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037).These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC.

Adenocarcinoma↗
PubMed2005

Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease.

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance caused by narrowing and loss of pulmonary microvasculature, which in its late stages becomes refractory to traditional therapies. We hypothesized that bone marrow-derived endothelial progenitor cells (EPCs), which normally function to repair and regenerate blood vessels, would restore pulmonary hemodynamics and increase microvascular perfusion in the rat monocrotaline (MCT) model of PAH. Mononuclear cells were isolated from the bone marrow of syngeneic Fisher-344 rats by Ficoll gradient centrifugation and cultured for 7 to 10 days in endothelial growth medium. Fluorescently labeled endothelial-like progenitor cells (ELPCs) engrafted at the level of the distal pulmonary arterioles and incorporated into the endothelial lining in the MCT-injured lung. The administration of ELPCs 3 days after MCT nearly completely prevented the increase in right ventricular systolic pressure seen at 3 weeks with MCT alone (31.5+/-0.95 versus 48+/-3 mm Hg, respectively; P<0.001), whereas injection of skin fibroblasts had no protective effect (50.9+/-5.4 mm Hg). Delayed administration of progenitor cells 3 weeks after MCT prevented the further progression of PAH 2 weeks later (ie, 5 weeks after MCT), whereas only animals receiving ELPCs transduced with human endothelial NO-synthase (eNOS) exhibited significant reversal of established disease at day 35 (31+/-2 mm Hg, P<0.005) compared with day 21 (50+/-3 mm Hg). Fluorescent microangiography revealed widespread occlusion of pulmonary precapillary arterioles 3 weeks after MCT, whereas arteriolar-capillary continuity and microvascular architecture was preserved with the administration of syngeneic ELPCs. Moreover, the delivery of ELPCs to rats with established PAH resulted in marked improvement in survival, which was greatest in the group receiving eNOS-transduced cells. We conclude that bone marrow-derived ELPCs can engraft and repair the MCT-damaged lung, restoring microvasculature structure and function. Therefore, the regeneration of lung vascular endothelium by injection of progenitor cells may represent a novel treatment paradigm for patients with PAH.

Animals↗
PubMed2005

A median sternotomy approach to right extrapleural pneumonectomy for mesothelioma.

Although pulmonary resections have been performed through median sternotomy, this approach for extrapleural pneumonectomy in the management of malignant pleural mesothelioma has not been described. We assessed the feasibility of a median sternotomy approach as an alternative to thoracotomy in right-sided resections. Over a 15-month period, this approach was attempted in 10 cases. In 7 of them, the entire procedure was completed without additional thoracotomy access. There were no postoperative deaths in this group. At median follow-up of 8 months, we have not encountered tumor progression in the scars.

Follow-Up Studies↗