Biomedical subjects

Philip T Nowicki

Publications and source records attributed to Philip T Nowicki.

10 recordsLinked to original sources

PubMed2007

Endothelial nitric oxide synthase in human intestine resected for necrotizing enterocolitis.

OBJECTIVE: To determine the expression and function of endothelial nitric oxide synthase (eNOS) in submucosal arterioles harvested from human intestine resected for necrotizing enterocolitis (NEC) or congenital bowel disease. STUDY DESIGN: eNOS expression was determined by using immunohistochemistry. The arteriolar diameter was measured in vitro at pressures of 10 to 40 mm Hg and also in response to the eNOS agonist acetylcholine (ACh), the exogenous nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine, and the smooth muscle relaxant papaverine. Arteriolar release of NO in response to ACh was determined with a Sievers NOAnalyzer. Hemodynamics were also determined at flow rates of 50 and 100 microL/min. RESULTS: eNOS was present in microvessels from both groups, but NEC arterioles failed to demonstrate physiological evidence of eNOS function: they constricted in response to pressure, failed to dilate or generate NO in response to ACh, and failed to dilate in response to flow. However, they dilated in response to exogenous NO and papaverine, indicating functional vascular smooth muscle and vasodilator reserve. CONCLUSION: eNOS-derived NO, a vasodilator in the newborn intestine, did not contribute to vasoregulation in arterioles harvested from intestine resected for NEC. These vessels were constricted; lack of eNOS-derived NO may contribute to this vasoconstriction.

Arterioles↗

PubMed2006

IL-1beta alters hemodynamics in newborn intestine: role of endothelin.

Studies were carried out to determine the effects of IL-1beta on newborn intestinal hemodynamics. IL-1beta increased the release of ET-1 by primary endothelial cells in a dose-dependent manner; as well, it reduced expression of the endothelin (ET) type B (ET(B)) receptor on endothelial cells and increased expression of the ET type A (ET(A)) receptor on vascular smooth muscle cells. IL-1beta increased endothelial cell endothelial nitric oxide (NO) synthase (eNOS) expression but did not enhance eNOS activity as evidenced by release of NO(x) into conditioned medium in response to acetylcholine or shear stress. The effects of IL-1beta on flow-induced dilation were evaluated in terminal mesenteric arteries in vitro. Pretreatment with IL-1beta (1 ng; 4 h) significantly attenuated vasodilation in response to flow rates of 100 and 200 microl/min. This effect was mediated, in part, by the endothelin ET(A) receptor; thus selective blockade of ET(A) receptors with BQ610 nearly restored flow-induced dilation. In contrast, exogenous ET-1 only shifted the diameter-flow curve downward without altering the percent vasodilation in response to flow. The effects of IL-1beta on ileal oxygenation were then studied using in vivo gut loops. Intramesenteric artery infusion of IL-1beta upstream of the gut loop caused ileal vasoconstriction and reduced the arterial-venous O(2) difference across the gut loop; consequently, it reduced ileal oxygenation by 60%. This effect was significantly attenuated by pretreatment with BQ610. These data support a linkage between the proinflammatory cytokine IL-1beta and vascular dysfunction within the intestinal circulation, mediated, at least in part, by the ET system.

Animals↗

PubMed2006

The effect of phenotype on mechanical stretch-induced vascular smooth muscle cell apoptosis.

The present study evaluated mechanical stretch-induced apoptosis in swine vascular smooth muscle cells (VSMC) of different phenotypes. We demonstrated that differentiated VSMC express a greater level of Bcl-2-associated death factor (BAD) and have a significant cell loss when exposed to mechanical stretch (10% elongation, 1 Hz) for 24 h. We further demonstrated that apoptosis was significantly increased only in differentiated VSMC exposed to mechanical stretch. To test the hypothesis that the intracellular level of BAD in VSMC determines its response to mechanical stretch-induced apoptosis, we examined whether BAD expression was upregulated by mechanical stretch-induced apoptosis and was associated with the increase in the apoptosis level of differentiated VSMC. When exposed to mechanical stretch, the expression of BAD in differentiated VSMC was elevated at 1 h and remained at higher levels during the application of stretch (24 h). In contrast, Bcl-2 expression was suppressed during the application of stretch. Moreover, the proapoptotic function of BAD was inhibited by overexpression of Bcl-2 through transient transfection of VSMC with pCEP4-Bcl-2 or incubation of VSMC with vascular epithelial growth factor. These results suggest that mechanical stretch-induced VSMC apoptosis is phenotype dependent. The higher levels of apoptosis of differentiated VSMC upon mechanical stretch were, at least in part, dependent on their intrinsic level of BAD.

Animals↗

PubMed2006

Intestinal O2 consumption in necrotizing enterocolitis: role of nitric oxide.

We tested the hypothesis that inducible isoform of nitric oxide synthase (iNOS)-derived nitric oxide (NO) inhibits oxygen consumption (VO2) in human intestine resected for necrotizing enterocolitis (NEC). Each NEC resection specimen was divided into two sections based on histologic appearance: healthy or diseased. Intestine removed from infants for reasons other than NEC was used as control. The tissue injury score (0-6, with 6 indicating complete necrosis) was 0.4 +/- 0.2 in control tissue, 1.2 +/- 0.4 in NEC-healthy tissue, and 4.6 +/- 0.5 in NEC-diseased tissue. Prominent iNOS staining was present in villus enterocytes in NEC-healthy tissue but not in the other tissue types. Intestinal VO2 (per direct oximetry, in nM O2/min/g) was significantly greater in control tissue than in NEC-healthy or NEC-diseased tissues. Accumulation of NO into buffer bathing intestinal slices (in nM NO/microL/g) was greater in NEC-healthy tissue than control or NEC-diseased tissues. The specific iNOS antagonist L-Nomega-(1-iminoethyl)-lysine (L-NIL) reduced buffer NO concentration 76% and increased VO2 by 90% in NEC-healthy tissue; however, L-NIL had no effect on NO or VO2 in control or NEC-diseased tissue. Addition of exogenous NO via S-nitroso-N-acetylpenicillamine depressed VO2 in NEC-healthy and control tissues but not NEC-diseased tissue. A significant correlation was present between buffer NO concentration and VO2 in NEC-healthy tissue. We conclude that iNOS-derived NO suppresses VO2 in intestine resected for NEC that demonstrates a relatively normal histology on light microscopy.

Animals↗

PubMed2005

Endothelin-1 in human intestine resected for necrotizing enterocolitis.

OBJECTIVES: We asked if the tissue concentration of the potent vasoconstrictor endothelin-1 (ET-1) is greater in areas of human preterm intestine that demonstrate histologic evidence of necrotizing enterocolitis (NEC) when compared with relatively healthy areas within the same resection specimen. We then evaluated if ET-1 participates in hemodynamic regulation within intestinal subserosal arterioles harvested from portions of human preterm intestine that demonstrate NEC. STUDY DESIGN: Human preterm intestine resected for NEC was divided into three zones based on proximity to the perforation (zone 1 most proximal, zone 3 most distal). Histologic evidence of NEC was determined in each zone (normal = 0, advanced necrosis = 6). The tissue concentration of ET-1 was determined by enzyme-linked immunosorbent assay within intestinal homogenates prepared from each zone. Arteriolar hemodynamics were determined in vitro on subserosal arterioles harvested from different zones. Arteriolar flow rate, diameter, and resistance were determined at pressure gradients (DeltaP) of 20 and 40 mmHg under control conditions and again after blockade of endothelin ET A receptors with BQ610 (10 -9 mol/L). RESULTS: The tissue concentration of ET-1 (pg/mg protein) and histologic score in the three zones were: zone 1: 84 +/- 14, 5.5 +/- 0.3; zone 2: 99 +/- 12, 4.7 +/- 0.4, and zone 3: 33 +/- 9, 0.8 +/- 0.6, respectively (M +/- SD, n = 10 resection specimens, P < .05, zone 3 vs zones 1 and 2). Zone 2 arterioles demonstrated significantly lower flow rate and diameter and increased resistance under control conditions than zone 3 arterioles when DeltaP was either 20 or 40 mmHg (n = 7, P < .05). Treatment with BQ610 had no effect on zone 3 arterioles but significantly vasodilated zone 2 arterioles, increasing flow rate and vessel diameter, and decreasing vascular resistance (n = 7, P < .05). CONCLUSIONS: The tissue concentration of ET-1 is greater in human preterm intestine that demonstrates histologic evidence of NEC. Arterioles harvested from intestine exhibiting histologic evidence of NEC demonstrate vasoconstriction when compared with arterioles from relatively healthy intestine in the same resection specimen. This vasoconstriction was reversed by blockade of endothelin ET A receptors.

Arterioles↗

PubMed2005

Ischemia and necrotizing enterocolitis: where, when, and how.

While it is accepted that ischemia contributes to the pathogenesis of necrotizing enterocolitis (NEC), three important questions regarding this role subsist. First, where within the intestinal circulation does the vascular pathophysiology occur? It is most likely that this event begins within the intramural microcirculation, particularly the small arteries that pierce the gut wall and the submucosal arteriolar plexus insofar as these represent the principal sites of resistance regulation in the gut. Mucosal damage might also disrupt the integrity or function of downstream villous arterioles leading to damage thereto; thereafter, noxious stimuli might ascend into the submucosal vessels via downstream venules and lymphatics. Second, when during the course of pathogenesis does ischemia occur? Ischemia is unlikely to the sole initiating factor of NEC; instead, it is more likely that ischemia is triggered by other events, such as inflammation at the mucosal surface. In this context, it is likely that ischemia plays a secondary, albeit critical role in disease extension. Third, how does the ischemia occur? Regulation of vascular resistance within newborn intestine is principally determined by a balance between the endothelial production of the vasoconstrictor peptide endothelin-1 (ET-1) and endothelial production of the vasodilator free radical nitric oxide (NO). Under normal conditions, the balance heavily favors NO-induced vasodilation, leading to a low resting resistance and high rate of flow. However, factors that disrupt endothelial cell function, eg, ischemia-reperfusion, sustained low-flow perfusion, or proinflammatory mediators, alter the ET-1:NO balance in favor of constriction. The unique ET-1-NO interaction thereafter might facilitate rapid extension of this constriction, generating a viscous cascade wherein ischemia rapidly extends into larger portions of the intestine.

Endothelin-1↗

PubMed2003

Presentation of nitric oxide regulates monocyte survival through effects on caspase-9 and caspase-3 activation.

In the absence of survival factors, blood monocytes undergo spontaneous apoptosis, which involves the activation of caspase-3. Although nitric oxide can block caspase-3 activation and promote cell survival, it can also induce apoptosis. We hypothesized that nitrosothiols that promote protein S-nitrosylation would reduce caspase-3 activation and cell survival, whereas nitric oxide donors (such as 1-propamine 3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA) NONOate and diethylamine (DEA) NONOate) that do not target thiol residues would not. Using human monocytes as a model, we observed that nitrosothiol donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine suppressed caspase-9 and caspase-3 activity and DNA fragmentation. In contrast, PAPA or DEA NONOate did not promote monocyte survival events and appeared to inhibit monocyte survival induced by macrophage colony-stimulating factor. The caspase-3-selective inhibitor DEVD-fluoromethyl ketone reversed DNA fragmentation events, and the caspase-9 inhibitor LEHD-fluoromethyl ketone reversed caspase-3 activity in monocytes treated with PAPA or DEA NONOate in the presence of macrophage colony-stimulating factor. These results were not caused by differences in glutathione levels or the kinetics of nitric oxide release. Moreover, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine directly blocked the activity of recombinant caspase-3, which was reversed by the reducing agent dithiothreitol, whereas PAPA or DEA NONOate did not block the enzymatic activity of caspase-3. These data support the hypothesis that nitrosylation of protein thiol residues by nitric oxide is critical for promoting the survival of human monocytes.

Caspase 3↗

PubMed2003

Development of the myogenic response in postnatal intestine: role of PKC.

Previous attempts to determine developmental changes in the vascular myogenic response have been confounded by the presence of competing vasoactive stimuli or the use of isolated vessels with markedly different baseline diameters. To circumvent these issues, small mesenteric arteries (diameter approximately 150 microm) from 1- and 10-day-old piglets were studied in vitro under no-flow conditions. In situ studies demonstrated that the intravascular pressure and diameter of these vessels were similar in both age groups, allowing an effective comparison of the myogenic response not obscured by differences in basal diameter. The pressure-diameter relationship was age specific. Thus, although small mesenteric arteries from both age groups demonstrated myogenic constriction in response to stepwise increases in pressure (0 to 100 mmHg, in 20-mmHg increments), the intensity of contraction was significantly greater in vessels from 1-day-old piglets particularly within the pressure range normally experienced by these vessels in situ. Attenuation or activation of PKC with calphostin C or indolactam, respectively, substantially altered the pressure-diameter relationship in 1-, but not 10-day-old arteries; thus calphostin C essentially eliminated the contractile response to pressure elevation in younger subjects, whereas indolactam significantly increased the intensity of the myogenic response and shifted its activation point to a lower pressure range. Immunoblots carried out on protein recovered from these arteries revealed the presence of alpha, beta, epsilon, iota, and lambda; notably, expression of the alpha- and epsilon-isoforms substantially decreased between postnatal days 1 and 10.

Animals↗

PubMed2002

Newborn intestinal circulation. Physiology and pathophysiology.

The physiologic characteristics of the newborn intestinal circulation are unique when compared with the adult condition. Most important, intestinal vascular resistance across newborn intestine is exceptionally low and this transient reduction is mediated by an increased constitutive and stimulated production of NO. The low vascular resistance characteristic of newborn intestine alters the capacity of this vasculature to respond to systemic circulatory perturbations, such as hypotension and arterial hypoxemia. The essential role of endothelial production of NO in maintaining newborn intestinal hemodynamics might be important in the pathogenesis of NEC, because endothelial dysfunction would limit, or possibly eliminate, NO production, leading to substantial intestinal ischemia.

Blood Circulation↗

PubMed2002

Developmental expression of eNOS in postnatal swine mesenteric artery.

Developmental changes in the expression of endothelial nitric oxide synthase (eNOS) within the mesenteric artery of swine were studied in fetal (110 days postconception/117 days total gestation) and on postnatal days 1, 3, 10, and 30. Subjects in the 1-day-old group were subdivided into fed and nonfed. Transcription of eNOS was determined by real-time PCR, protein expression was evaluated by Western blotting, and hemodynamic and oxygenation parameters were measured within in situ gut loops before and after the administration of N(G)-monomethyl-L-arginine (L-NMMA). The abundance of eNOS mRNA remained steady throughout all ages. In contrast, expression of eNOS protein was twofold greater in the 1-day-old fed subjects compared with fetal or 1-day-old nonfed subjects. eNOS protein expression remained elevated on day 3, increased on day 10, and then declined to a level similar to the day 1 nonfed group by postnatal day 30. Intestinal vascular resistance was 31% lower in the day 1 fed group when compared to the day 1 nonfed group; resistance continued to decline through day 10 but then significantly increased on day 30. We conclude that the expression of eNOS changes within the mesenteric artery during early postnatal development at a posttranscriptional level.

Aging↗