Evidence for the nitric oxide pathway as a potential mode of action in fenoldopam-induced vascular injury.

Fenoldopam, a dopaminergic DA1 agonist, induces vasodilatation via nitric oxide (NO), and this may be associated with mesenteric arterial injury. NO is produced from the enzymatic action of nitric oxide synthase (NOS), which is regulated by the shear-stress mediating protein caveolin-1. Profound vasodilatation and accompanied decreased shear are early events that could initiate vascular injury. Therefore, it is of interest to determine the role of caveolin-1 and the NO pathway in fenoldopam-induced vascular injury. At sites of fenoldopam-induced mesenteric arterial injury, decreased caveolin-1 expression and apoptosis were prominent immunohistochemical findings. An additional finding at these sites of injury were loss and/or reduced expression of caveolin-1 regulated structural proteins, connexin-43, (gap junction) ZO-1, and claudin (tight junctions). Because functional loss of caveolin-1 is associated with increased NOS activity and vasodilatation via NO, studies were conducted to show a NO donor produced vascular lesions in the mesenteric arteries morphologically similar to those induced by fenoldopam. Moreover, the incidence and severity of fenoldopam-induced vascular injury were reduced when an NOS inhibitor or a scavenger of NO-generated free radicals were coadministered with fenoldopam. Collectively, these data suggest that caveolin-1 and its regulated NO pathway may play an important role in vasodilatory drug-induced vascular injury.

Potential candidate genomic biomarkers of drug induced vascular injury in the rat.

Drug-induced vascular injury is frequently observed in rats but the relevance and translation to humans present a hurdle for drug development. Numerous structurally diverse pharmacologic agents have been shown to induce mesenteric arterial medial necrosis in rats, but no consistent biomarkers have been identified. To address this need, a novel strategy was developed in rats to identify genes associated with the development of drug-induced mesenteric arterial medial necrosis. Separate groups (n=6/group) of male rats were given 28 different toxicants (30 different treatments) for 1 or 4 days with each toxicant given at 3 different doses (low, mid and high) plus corresponding vehicle (912 total rats). Mesentery was collected, frozen and endothelial and vascular smooth muscle cells were microdissected from each artery. RNA was isolated, amplified and Affymetrix GeneChip® analysis was performed on selectively enriched samples and a novel panel of genes representing those which showed a dose responsive pattern for all treatments in which mesenteric arterial medial necrosis was histologically observed, was developed and verified in individual endothelial cell- and vascular smooth muscle cell-enriched samples. Data were confirmed in samples containing mesentery using quantitative real-time RT-PCR (TaqMan™) gene expression profiling. In addition, the performance of the panel was also confirmed using similarly collected samples obtained from a timecourse study in rats given a well established vascular toxicant (Fenoldopam). Although further validation is still required, a novel gene panel has been developed that represents a strategic opportunity that can potentially be used to help predict the occurrence of drug-induced mesenteric arterial medial necrosis in rats at an early stage in drug development.

The use of in situ perfusion of the rat mesentery as a model to investigate vascular injury directly induced by drugs.

Exposure of the vasculature to vasodilators, pharmaceuticals and industrial chemicals may lead to injury of the blood vessel wall in animals. Vascular injury may begin with changes in the permeability of vascular endothelial cell and vessels, resulting in possible hemorrhage and edema leading subsequently to immune cell infiltration. The present study was undertaken to determine if the direct exposure of the Sprague Dawley rat mesenteric vasculature through the perfusion of aminophylline, fenoldopam, compound 48/80, histamine or serotonin has any such effects on the blood vessels, and if the two vital dyes Monastral blue B and Evans blue can be used to enhance the visualization of the vascular damage. Microscopic visualization was enhanced by the use of dyes and a variety of alterations of the perfused mesenteric vessels were detected, including varying degrees of mast cell degranulation, microvascular vasodilatation and increased vascular permeability. Macroscopic evidence of vascular damage was minimal. This study demonstrates that in situ perfusion of the rat mesentery is a simple and useful method to eliminate the influence of a variety of physiologic influences or homeostatic responses and can be used to further investigate drug-induced vascular damage.

Transcriptional profiling of laser capture microdissected rat arterial elements: fenoldopam-induced vascular toxicity as a model system.

Transcriptional profiling of specific elements of vasculature from animal models of vascular toxicity is an approach to gain insight into molecular mechanisms of vascular injury. Feasibility of using laser capture microdissection (LCM) to evaluate differential gene expression in selected elements of mesenteric arteries (MA) from untreated rats and rats given a single vasotoxic dose of 100 mg/kg Fenoldopam and euthanized 1 or 4 hours postdose was assessed. Regions of MA (endothelial cells [EC] and vascular smooth muscle cells [VSMC]) were selectively microdissected from optimal-cutting-temperature (O.C.T.)-embedded-frozen tissue sections. RNA was isolated, linearly amplified (LA), and hybridized to Affymetrix GeneChips. Enrichment for specific vascular elements was evident by unique gene-expression profiles. Statistical analysis indicated that Fenoldopam treatment resulted in differential expression of 333 versus 458 genes in EC and 371 versus 618 genes in VSMC at the 1-hour or 4-hour time point, respectively. Analysis of regulated EC and VSMC genes common to both time points identified several gene functions or pathways affected by treatment. Several genes were identified in EC and/or VSMC that have not been previously linked to vascular structure or function. These data indicate that tissue-element-enrichment by LCM in conjunction with LA and GeneChip analysis offers a refined approach for assessment of injury-mediated transcriptome changes in distinct elements of the vasculature.

Immunohistochemical study on inducible type of nitric oxide (iNOS), basic fibroblast growth factor (bFGF) and tumor growth factor-beta1 (TGF-beta1) in arteritis induced in rats by fenoldopam and theophylline, vasodilators.

Arteritis induced in rats by vasodilators, fenoldopam and theophylline, was examined immunohistochemically for expressions of inducible type of nitric oxide synthase (iNOS), basic fibroblast growth factor (bFGF) and tumor growth factor-beta1 (TGF-beta1). Rats were administered fenoldopam for 24 hours by intravenous infusion with or without following repeated daily oral administrations of theophylline. Irrespective of theophylline administration, iNOS antigens were remarkably abundant in ED-1-positive cells on day 5 and 8 post-fenoldopam-infusion (DPI); bFGF antigens were remarkably abundant in ED-1-positive cells on 1 and 3 DPI; TGF-beta1 antigens were observed in ED-1-positive cells on and after 5 DPI. These results suggest that the peak expression of iNOS antigen was followed by that of bFGF antigen, and bFGF may have a suppressive effect on iNOS expression in these rat arteritis models. On the other hand, TGF-beta1 was not considered to have a suppressive effect on iNOS expression in these models.

Histopathological and immunohistochemical studies on arteritis induced by fenoldopam, a vasodilator, in rats.

Fenoldopam, a dopaminergic (DA1) agonist, has been reported to induce medial necrosis and adventitial inflammatory response in the splanchnic arteries in rats. This study was carried out to clarify the detailed time course of the inflammatory responses, using antibodies for the inflammatory cell markers, CD3 (T cell), CD20 (B cell) and ED-1 (macrophage), and inflammatory serum factors, IgG, IgM and C3. Rats were administered fenoldopam for 24 hours by intravenous infusion. Histopathologically, medial necrosis with hemorrhage was observed at the end of infusion, but it almost disappeared on day 7 post-infusion. Adventitial inflammatory responses with ED-1-, CD3- and CD20-positive cells were very slight at the end of infusion, became prominent with marked fibrosis on days 3 and 5, decreased on day 7, and subsided on day 14. The serum factors were first present in the area of medial necrosis, then shifted to the subendothelial space or cytoplasm of smooth muscle cells, and disappeared on day 14 post-infusion. Gaps in the external elastic lamina were observed on days 3 and 5 post-infusion, and IgG and IgM were present outside the gaps in the adventitia. These results provided us with more detailed information on the inflammatory responses following medial damage induced by vasodilators.

Evaluation of plasma von Willebrand factor as a biomarker for acute arterial damage in rats.

Plasma von Willebrand factor (vWF) was evaluated as a potential biomarker of acute arterial damage in rats after a vasotoxic dose of the dopaminergic vasodilator, fenoldopam (FP). Male Sprague-Dawley rats were given FP or isotonic saline by subcutaneous injection, and plasma vWF was measured at 2, 6, and 24 hours after challenge. Mean plasma vWF values increased in FP-treated rats compared to controls at 2 hours (167 vs 122%; p < 0.05) and 6 hours postdose (172 vs 130%; p < 0.01) but were comparable to control values after 24 hours. Mesenteric arterial lesions were observed microscopically in all FP-treated rats 24 hours postdose but were not present in rats at 1, 2, 4, 6, or 8 hours after FP challenge. Further, plasma vWF concentrations increased in saline-treated rats after only the minimal perturbation of repeated venipuncture. These results indicate an early, minimal, and transient release of vWF that precedes the onset of morphologically evident vascular damage. The minimal increases in plasma vWF concentrations were of limited predictive value, may be more reflective of an acute-phase reactant response, and were not considered a reliable biomarker of acute FP-induced arterial damage in the rat.

Arterial lesions induced by phosphodiesterase III (PDE III) inhibitors and DA(1) agonists.

Compounds that inhibit the low Km, cGMP-inhibitable form of phosphodiesterase (type III) and the DA(1) agonist, fenoldopam, are potent vasodilators that have been associated with segmental medial haemorrhagic necrosis in susceptible arterial beds following administration of suprapharmacological doses to dogs and/or rats. Morphological and haemodynamic investigative studies with PDE III inhibitors support the hypothesis that the arterial toxicity is the consequence of the vasodilator pharmacology of these compounds. Investigative data also suggest that similar mechanisms are involved in the pathogenesis of arterial lesions induced by fenoldopam and the K(+) channel opener, minoxidil.

Characterization of the arteritis induced by infusion of rats with UK-61,260, an inodilator, for 24 h. A comparison with the arteritis induced by fenoldopam mesylate.

Administration of fenoldopam mesylate (FM), a dopaminergic agonist, or of cyclic cAMP phosphodiesterase inhibitors (PDE III), for example theophylline and caffeine, induces arteritis in the rat. In this study we characterized the arteritis induced by UK-61,260, an investigational inotropic agent with vasodilatory properties which displays an inhibitory action on cyclic AMP phosphodiesterase, in comparison with lesions induced by FM. The compounds were administered to Sprague-Dawley rats by intravenous infusion over 24 h (FM and UK-61,260), orally or subcutaneously (UK-61,260); the rats were killed and necropsied for pathological examination at various times between 0 h and 28 days post-infusion. Infusion of UK-61,260 at doses of 100, 300 or 400 mg/kg produced arteritis mainly in the mesenteric arteries and occasionally in the renal, pancreatic, gastric and coronary arteries. There were no arterial lesions after infusion of 30 mg/kg, or after administration of 30, 100 or 200 mg/kg per day subcutaneously for 7 days, or after acute administration of 100, 300, 400 or 600 mg/kg orally. Infusion of rats with 72 or 144 mg/kg FM produced arteritis over a wider range of tissues than did UK-61,260. However, the arterial lesions produced by infusion of either drug have the same initial aspect and a similar evolution with time. Immediately after the end of the infusion, minimal necrosis and haemorrhage occurred in the media only, without involvement of the endothelium or the perivascular space. This indicates that the media of the artery is the primary site of injury. The lesions seen 1 and 3 days post-infusion were characterized by severe medial necrosis and haemorrhage with perivascular acute inflammation and appeared macroscopically as haemorrhagic spots on the vessels. On days 7, 14 and 28 post-infusion, no medial necrosis or haemorrhage were present, while perivascular chronic inflammation and moderate smooth muscle hyperplasia were seen. It appeared, therefore, that the lesions underwent repair in 28 days, but footprints of the damage were still present 28 days post-infusion. The similarity between arteritis induced in rats by fenoldopam or by UK-61,260, at doses inducing PDE III inhibition, is consistent with the view that they have a similar pathogenesis. In our view it is probable that these pharmacologically and chemically distinct drugs trigger an increase in intracellular levels of cAMP which in turn triggers vascular damage. The arterial changes observed in the current study after acute administration may explain the increased incidence of polyarteritis nodosa occurring in long term toxicity studies with FM or PDE III inhibitors.