Adel Giaid

Localization of Urotensin-II Immunoreactivity in Normal Human Kidneys and Renal Carcinoma

Human urotensin-II (U-II) is a cyclic 11-amino-acid residue peptide with a wide range of vasoactive properties dependent on the anatomic site and the species studied. The purpose of this study was to determine the localization of human U-II in normal human kidneys and in renal carcinoma. Normal human kidneys (n=11) and eight cases of clear-cell carcinoma were immunostained with a polyclonal antibody to human U-II. In normal human kidneys, U-II was mostly present in the epithelial cells of tubules and ducts, with greater intensity in the distal convoluted tubules. Moderate U-II immunoreactivity was seen in the endothelial cells of renal capillaries, but only focal immunoreactivity was found in the endothelial cells of the glomeruli. No staining was found in the veins. All tumors expressed moderate U-II immunoreactivity in the cancer cells and vasculature. Here we demonstrate abundant expression of U-II in normal human kidneys and renal carcinoma. These findings suggest that the vasoactive and growth-mediator peptide U-II may contribute to the pathophysiology of the human renal system.

Role of urotensin II in health and disease

Urotensin II (UII) is an 11 amino acid cyclic peptide originally isolated from the goby fish. The amino acid sequence of UII is exceptionally conserved across most vertebrate taxa, sharing structural similarity to somatostatin. UII binds to a class of G protein-coupled receptor known as GPR14 or the urotensin receptor (UT). UII and its receptor, UT, are widely expressed throughout the cardiovascular, pulmonary, central nervous, renal, and metabolic systems. UII is generally agreed to be the most potent endogenous vasoconstrictor discovered to date. Its physiological mechanisms are similar in some ways to other potent mediators, such as endothelin-1. For example, both compounds elicit a strong vascular smooth muscle-dependent vasoconstriction via Ca(2+) release. UII also exerts a wide range of actions in other systems, such as proliferation of vascular smooth muscle cells, fibroblasts, and cancer cells. It also 1) enhances foam cell formation, chemotaxis of inflammatory cells, and inotropic and hypertrophic effects on heart muscle; 2) inhibits insulin release, modulates glomerular filtration, and release of catecholamines; and 3) may help regulate food intake and the sleep cycle. Elevated plasma levels of UII and increased levels of UII and UT expression have been demonstrated in numerous diseased conditions, including hypertension, atherosclerosis, heart failure, pulmonary hypertension, diabetes, renal failure, and the metabolic syndrome. Indeed, some of these reports suggest that UII is a marker of disease activity. As such, the UT receptor is emerging as a promising target for therapeutic intervention. Here, a concise review is given on the vast physiologic and pathologic roles of UII.

Immunohistochemical localization of nitric oxide synthase and the oxidant peroxynitrite in lung transplant recipients with obliterative bronchiolitis.

BACKGROUNDnObliterative bronchiolitis (OB) is a disease affecting a large percentage of lung and heart-lung transplant recipients. Histologically, the disease is characterized by inflammation, cellular proliferation, and obliteration of terminal airways.nnnMETHODSnWe investigated the production of inducible and constitutive nitric oxide synthases and peroxynitrite by immunohistochemistry in the lungs of control subjects (n=14) compared with those of transplant recipients with OB (n=8).nnnRESULTSnStrong immunoreactivity for inducible nitric oxide synthase and nitrotyrosine, a marker of protein nitration by peroxynitrite, was seen in inflammatory cells, airway epithelium, and vascular endothelium of patients with OB, compared with little immunoreactivity in control lungs. Immunoreactivity for constitutive nitric oxide synthase was abundant in the airway epithelium and vascular endothelium of control lungs, however, it was decreased in airway epithelial cells and arterial endothelial cells of patients with OB.nnnCONCLUSIONSnWe conclude that increased formation of the potent oxidant peroxynitrite and decreased production of endothelial nitric oxide may contribute to the functional and morphological abnormalities of OB.

Chemokine- and cytokine-induced expression of endothelin 1 and endothelin-converting enzyme 1 in endothelial cells

BACKGROUNDnEndothelin 1 (ET-1) is a product of endothelial and many other cell types that possesses a wide range of actions, including vasoconstriction, bronchoconstriction, and mitogenic activity on smooth muscle cells and fibroblasts. ET-1 release and expression is induced in several disease conditions associated with inflammation and cellular injury.nnnOBJECTIVEnThe purpose of this study is to determine the effects of alpha-chemokines (IL-8 and melanoma growth-stimulating activator), beta-chemokines (monocyte chemotactic protein 1, macrophage inflammatory protein 1alpha [MIP-1alpha], MIP-1beta, and RANTES), and proinflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) on the expression of both ET-1 and endothelin-converting enzyme 1 (ECE-1) by human umbilical vein endothelial cells.nnnMETHODSnSubconfluent monolayers of human umbilical vein endothelial cells were incubated with each chemokine individually for 24 hours or with a mixture (cytomix) of TNF-alpha, IL-1beta, and IFN-gamma for 6 and 24 hours.nnnRESULTSnIncubation with the alpha-chemokines melanoma growth-stimulating activity and IL-8 did not significantly increase ET-1 and ECE-1 messenger (m)RNA expression and had no effect on ET-1 release. Monocyte chemotactic protein 1 exerted the most potent increase in ET-1 and ECE-1 mRNA and ET-1 release among all chemokines studied (P <.05). MIP-1alpha and RANTES exerted a moderate, but significant, increase on the ET system (P <.05). The cytomix resulted in a significant increase in ET-1 and ECE-1 mRNA expression (P <.05).nnnCONCLUSIONnThese data demonstrate that, like cytokines, chemokines can induce endothelial ET-1 and ECE-1 in vitro and suggest a possible role for these inflammatory mediators in the induction of the ET system in inflammatory and vascular diseases.

Expression of endothelin-1 and endothelin-converting enzyme-1 mRNAs and proteins in failing human hearts.

Accumulating evidence suggests that endogenous endothelin-1 (ET-1) may contribute to the development of heart failure. In this study we determined sites of ET-1 synthesis and production in the failing human myocardium by immunohistochemistry and in situ hybridization for ET-1 and endothelin-converting enzyme-1 (ECE-1). Myocardial tissues were obtained from 19 patients with heart failure and from four noncardiac patients as controls. In both failing and nonfailing hearts, apparent immunoreactivity for ET-1 and ECE-1 was consistently seen in cardiac myocytes. Endothelial cells of intramyocardial coronary arteries and veins had only weak or focal ET-1 and apparent ECE-1 immunoreactivities. On the other hand, in situ hybridization showed strong signals for ET-1 and ECE-1 mRNAs in vascular endothelial cells but a lesser intensity of signals in cardiac myocytes. Apparent immunoreactivity and strong hybridization signals for both ET-1 and ECE-1 were seen in macrophages, which were abundant in infarcted regions of ischemic cardiomyopathy and in myocardium of septic patients but were rare in healthy hearts. These results suggest that, in failing human heart, vascular endothelial cells and macrophages rather than cardiac myocytes appear to be the principal ET-1 synthetic sites, although ET-1 peptides are abundantly present in cardiac myocytes of both failing and nonfailing hearts. Endogenous ET-1 may play a pathophysiologic role in human heart failure.

Potential role of endothelin-1 in pulmonary fibrosis: from the bench to the clinic.

Endothelin-1 (ET-1) plays a central role in lung fibrosis. It is released in the lung at low concentrations from the endothelium, epithelium, and vascular smooth muscle cells and orchestrates a variety of effects. In the context of wound healing, ET-1 acts with other profibrotic mediators to recruit fibroblasts and allow for their differentiation to contractile myofibroblasts. These specialized cells in turn lay down fibrotic tissue and contract at the site of lesions to restore tissue integrity. Apoptosis and reversion to quiescence ensues. However, in diseases of the lung such as idiopathic pulmonary fibrosis (IPF), the fibrotic response is uncontrolled. Progressive injury to lung tissue, isolated both temporally and geographically, is uncontrolled and eventually causes enough tissue damage to alter pulmonary architecture and compromise function. The initiating mechanisms are as of yet largely unknown; however, ET-1 has clearly emerged as a key mediator of this disease. Here, a comprehensive overview of the role of ET-1 in fibrosis is given. A guided perspective begins from the scope of its various molecular interactions to its many cellular processes, and finally to the implications of these functions in IPF.

A novel charcoal-induced model of obliterative bronchiolitis–like lesions: Implications of chronic nonspecific airway inflammation in the development of posttransplantation obliterative bronchiolitis

OBJECTIVESnIn this study, we describe the development of a nonallogeneic animal model of obliterative bronchiolitis-like lesions. Furthermore, we examined whether chronic rejection alone can lead to the development of obliterative bronchiolitis or whether additional nonspecific airway inflammation is required.nnnMETHODSnPart I: Rats were intratracheally injected with 0.2 ml of activated charcoal or sorbitol solution (carrier for charcoal control). Animals were put to death beginning at 2 weeks up to 20 weeks. Part II: Animals were divided into three groups: group I, underimmunosuppressed Brown Norway to Lewis lung allografts; group II, charcoal-treated underimmunosuppressed allografts; and group III, charcoal-treated rats. Animals were put to death at 3 months after transplantation.nnnRESULTSnPart I: In charcoal-laden bronchioles, subacute nonspecific airway inflammation was detected at 2 weeks. Slow, subclinical fibroproliferation ensued during the following weeks. Obliterative bronchiolitis-like lesions were observed in 80% of charcoal-treated animals at 12 weeks. Part II: Allografts developed extensive vascular lesions consistent with acute and chronic vascular rejection. Obliterative bronchiolitis-like lesions were scarcely detected. Charcoal-treated allografts demonstrated evidence of diffuse and severe obliterative bronchiolitis-like lesions.nnnCONCLUSIONSnTranstracheal injection of activated charcoal into native lungs results in slowly progressive airway injury and inflammation leading to obliterative airway lesions. Inadequate immunosuppression primarily results in chronic vascular rejection but not obliterative bronchiolitis. Underimmunosuppressed allografts subjected to nonspecific airway inflammation develop obliterative airway lesions that are more prominent than in native lungs. This suggests that a cofactor to chronic rejection is likely necessary for the development of lung transplant obliterative bronchiolitis.

Detection of human probombesin mRNA in neuroendocrine (small cell) carcinoma of the lung. In situ hybridization with cRNA probe.

The production of human bombesin (gastrin‐releasing peptide), a peptide with mitogenic action, is a recognized feature of neuroendocrine (small cell) carcinoma of the lung. However, immunostaining of bombesin is not always possible in these tumors, probably because of poor storage mechanisms or rapid release of hormone. Molecular biological analysis of the gene encoding human bombesin has revealed the DNA sequence of human pro‐bombesin. We have used in situ hybridization to study the expression of the human bombesin gene at the cellular level in small cell carcinoma of the lung. Probombesin cDNA was subcloned in pSP64 vector, linearized with Bam HI and transcribed in the presence of phosphorus 32 (32P)‐cytosine triphosphate (CTP) and SP6 polymerase. The cRNA probe was applied to tissue sections (from six cases of small cell carcinoma of the lung, freshly fixed in 4% paraformaldehyde), cell culture preparations (two different cell lines of small cell carcinoma), and cytologic specimens (smears of cells from three different cases of small cell carcinoma). Hybridization of probombesin mRNA was detected in tumor cells in all samples. Specificity of the signal was determined by control experiments, including the use of a probe which has a sequence identical to probombesin mRNA. Our results provide evidence for the expression of the bombesin gene in small cell carcinoma of the lung at a cellular level and show that probombesin mRNA is highly expressed in these tumors.

Immunohistochemical characterization of inflammatory and proliferative events during chronic rejection in rat lung allografts.

BACKGROUNDnChronic rejection is assumed to be the principle cause of airway injury leading to obliterative bronchiolitis (OB) after lung transplantation (Tx). To better understand the contribution of chronic rejection in the development of OB in allografted lungs, we examined the histopathological changes and cytokine expression in inadequately immunosuppressed rat lung allografts.nnnMETHODSnThree groups of rats were studied: group I, control nontransplanted Lewis (Lew) rats (n=5); group II, syngeneic Lew-to-Lew isografts (n=25); and group III, Brown Norway-to-Lew allografts (n=25). Groups II and III received two single doses of cyclosporine on postoperative days 2-3. Transplanted animals were killed (n=5) at monthly intervals from 2 months to 6 months after Tx. Resected lungs were stained with hematoxylin and eosin, Massons trichrome, and Van Giesons elastin, and immunostained with antisera to interleukin (IL)-1beta, IL-8, and basic fibroblast growth factor (bFGF). The intensity of immunostaining was graded from 0 to 4 (0=no staining, 4=strong staining).nnnRESULTSnIn groups I and II, normal airways and vessels were observed. Minimal intensity and distribution of immunostaining for all markers were detected in groups I and II. Group III allografts demonstrated acute grade II-III vascular rejection with mild bronchiolar injury and inflammation at 2 months after Tx. At 6 months after Tx, all allografts demonstrated severe and diffuse chronic vascular rejection. Late airway changes consistent with OB were detected in four of five allografts, however, these lesions were expressed infrequently. Immunohistochemical findings revealed moderate to strong expression for IL-8 and bFGF over the airway epithelium, acute and chronic inflammatory cells, and fibroblasts in allografts at 2 months after Tx. Despite focal development of OB at 6 months, intensity and distribution of immunostaining significantly decreased for all three cytokine markers.nnnCONCLUSIONSnInadequate immunosuppression of rat lung allografts leads primarily to chronic vascular rejection but fails to induce severe and diffuse development of OB. In this animal model, cytokines IL-1beta, IL-8, and bFGF are likely to play an important role in the early inflammatory phase but not during the late proliferative events of chronic rejection.

Effect of endothelin receptor antagonist on lung allograft apoptosis and NOSII expression

BACKGROUNDnIt is postulated that apoptosis contributes to ischemia-reperfusion graft dysfunction after lung transplantation. The purpose of this study was to determine whether the improvement in lung function that we previously observed with the use of an endothelin-1 (ET-1) receptor antagonist after ischemia-reperfusion injury is associated with a reduction in inducible nitric oxide synthase (NOSII) expression and programmed cell death.nnnMETHODSnLeft lung canine allotransplantation was performed. Harvested lung blocks were preserved with modified Eurocollins solution and stored at 4 degrees C for 18 to 20 hours. Lung allografts were tested for the expression of NOSII by immunohistochemistry, and extent of apoptosis by terminal dUTP nick end-labeling (TUNEL). Animals blindly received either an intravenous infusion of saline (control) or the ET-1 receptor antagonist (SB209670) (15 microg/kg/min). Infusion began 30 minutes pretransplantation and continued to 6 hours posttransplantation.nnnRESULTSnImmunohistochemical analysis demonstrated significantly stronger NOSII immunostaining in the allografts of the saline control group (36.5%+/-3.6%) compared with native right lungs (6.9%+/-1.3%, p < 0.001) or the ET-receptor antagonist treatment group (9.6%+/-1.4%, p < 0.001). The TUNEL staining revealed a significantly stronger labeling in the allografts of the saline treatment control group (40.7%+/-6.2%) compared with native right lungs (5.0%+/-0.6%, p < 0.005) or the ET receptor antagonist treatment group (14.1%+/-2.8%, p < 0.01).nnnCONCLUSIONSnWe conclude that treatment of lung allografts with the ET-1 receptor antagonist SB209670 reduces the area of NOSII expression and the extent of apoptosis, factors known to contribute to the process of prolonged ischemia-reperfusion injury.