Calvert Louden

Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14

Urotensin-II (U-II) is a vasoactive ‘somatostatin-like’ cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 (refs 4, 5) and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

A role for endogenous endothelin-1 in neointimal formation after rat carotid artery balloon angioplasty. Protective effects of the novel nonpeptide endothelin receptor antagonist SB 209670.

The observation that levels of the mitogenic peptide endothelin-1 are elevated in the human coronary sinus after percutaneous transluminal coronary angioplasty (PTCA) has implicated endothelin-1 in the etiology of vascular restenosis. The present study examined this hypothesis in both an in vitro and an in vivo rat model of neointimal formation by using the novel nonpeptide endothelin receptor antagonist SB 209670. In vitro, endothelin-1 (1 nmol/L) induced a ninefold increase in rat aortic vascular smooth muscle [3H]thymidine incorporation. This endothelin A receptor-mediated effect was completely inhibited by SB 209670 (IC50, 6.2 +/- 2.2 nmol/L). In vivo, acute intra-arterial administration of exogenous endothelin-1 (5 to 500 pmol/kg over a 30-minute period immediately after angioplasty) dose-dependently augmented the degree of neointimal formation (by up to 150% when assessed 14 days after surgery). This response was evident as early as 7 days after angioplasty. Hemodynamic studies indicated that this action was unrelated to a systemic pressor action of the peptide. Administration of SB 209670 (2.5 mg/kg IP, twice a day for 3 days before and for 2 weeks after surgery) reduced neointimal formation by approximately 50% relative to control animals. Thus, the data indicate for the first time that (1) endothelin-1 promotes neointimal formation in vivo and (2) endogenous endothelin-1 is involved in the pathogenesis of angioplasty-induced lesion formation in the rat. Endothelin receptor antagonists such as SB 209670 may therefore serve as useful adjuncts to PTCA, attenuating the degree of vascular restenosis observed after vascular wall injury.

Delayed Expression of Osteopontin after Focal Stroke in the Rat

Focal brain ischemia induces inflammation, extracellular matrix remodeling, gliosis, and neovascularization. Osteopontin (OPN) is a secreted glycoprotein that has been implicated in vascular injury by promoting cell adhesion, migration, and chemotaxis. To investigate the possible involvement of OPN in brain matrix remodeling after focal stroke, we examined the expression of OPN in ischemic cortex after permanent or temporary occlusion of the middle cerebral artery (MCAO) of the rat. OPN mRNA and protein levels in nonischemic cortex were not detected consistently, although significant induction of OPN was observed in the ischemic cortex. OPN mRNA increased 3.5-fold at 12 hr and reached peak levels 5 d (49.5-fold; p < 0.001) after permanent MCAO. The profile of OPN mRNA induction after transient MCAO (160 min) with reperfusion was essentially the same as that of permanent MCAO. In situ hybridization and immunohistochemical studies demonstrated strong induction of OPN in the ischemic cortex, which was localized primarily in a subset of ED-1-positive macrophages that accumulated in the ischemic zone. Moreover, OPN immunoreactivity was detected in the matrix of ischemic brain, suggesting a functional role of the newly deposited matrix protein in cell–matrix interactions and remodeling. Indeed, using a modified Boyden chamber, we demonstrated a dose-dependent chemotactic activity of OPN in C6 astroglia cells and normal human astrocytes. Taken together, these data suggest that the upregulation of OPN after focal brain ischemia may play a role in cellular (glia, macrophage) migration/activation and matrix remodeling that provides for new matrix–cell interaction.

Expression of Endothelin-1, Endothelin-3, Endothelin-Converting Enzyme-1, and Endothelin-A and Endothelin-B Receptor mRNA After Angioplasty-Induced Neointimal Formation in the Rat

Endothelins (ETs) are potent vasoconstrictors known to play a role in tissue remodeling after vascular wall injury. The molecular mechanisms for the expression and functions of ETs and their receptors after carotid artery angioplasty are not fully understood. Using quantitative reverse transcription and polymerase chain reaction, the present study demonstrates the temporal mRNA expression of ET-converting enzyme-1 (ECE-1), preproET-1, preproET-3, and both ETA and ETB receptors after rat carotid artery balloon angioplasty. A significant increase in ECE-1 mRNA was observed at 6 hours (1.8-fold increase over control, P < .01) and 24 hours (1.7-fold increase, P < .01) in carotid arteries after angioplasty. In contrast, a significant increase in preproET-1 mRNA levels was not observed until 3 days (1.9-fold increase, P < .05) and 7 days (2.1-fold increase, P < .05). A similarly delayed increase in preproET-3 mRNA was observed at 7 days (2.8-fold increase, P < .05) and 14 days (2.6-fold increase, P < .05) after angioplasty. A parallel but marked increase in ETA and ETB receptor mRNAs compared with preproET-1 and -3 messages was observed after angioplasty. The levels of ETA receptor mRNA were elevated 29.3-fold (P < .001) and 24.3-fold (P < .01) at 3 and 7 days, respectively, after angioplasty. The increase in ETB receptor mRNA occurred slightly earlier than the increase in ETA receptor mRNA, showing 15.1-fold increase at 1 day (P < .001) and 11.3-fold increase at 3 days (P < .01) after angioplasty. Immunohistochemical studies using anti-ET antibodies demonstrated a corresponding increase in ET immunoactivity, which was distributed mainly in the neointimal cells 14 days after angioplasty. The increases in ECE-1, ET-1, and ET-3 and their receptor expression after balloon angioplasty suggest that these proteins play an active role in the pathogenesis of neointimal formation.

Loss of α-hemoglobin–stabilizing protein impairs erythropoiesis and exacerbates β-thalassemia

Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free α- and β-Hb subunits, which are unstable and cytotoxic. The α-Hb–stabilizing protein (AHSP) is an erythroid protein that specifically binds α-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free α-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP–/– erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable α-Hb, AHSP–/– erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by α-Hb in solution. Finally, loss of AHSP worsened the phenotype of β-thalassemia, a common inherited anemia characterized by excess free α-Hb. Together, the data support a model in which AHSP binds α-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in β-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of β-thalassemia in humans.

Osteopontin Expression in Platelet-Derived Growth Factor–Stimulated Vascular Smooth Muscle Cells and Carotid Artery After Balloon Angioplasty

Osteopontin (OPN), an arginine-glycine-aspartate (RGD)-containing adhesive glycoprotein, is constitutively expressed in rat aorta and carotid arteries and is markedly elevated in response to vascular injury. OPN is chemotactic for vascular smooth muscle cells (SMCs), suggesting a role in vascular remodeling. However, the mechanism for the regulation of OPN expression is poorly understood. In the present study, the effect of platelet-derived growth factor (PDGF) on OPN mRNA expression was investigated in cultured rat aortic SMCs (RASMCs). When RASMCs were stimulated with 1 nmol/L PDGF, a 2.4-fold increase in OPN mRNA expression was observed at 3 hours (P < .05) that peaked at 14 hours with a 6.7-fold increase (P < .001). This induction was blocked by a monoclonal anti PDGF antibody. Further studies revealed that OPN mRNA expression was induced by PDGF-AB or PDGF-BB but not by PDGF-AA, indicating that only the beta-type PDGF receptor mediates this response. Compared with basic fibroblast growth factor, epidermal growth factor, transforming growth factor-beta, and interleukin-1 beta, PDGF was the most potent factor studied to induce OPN mRNA expression in RASMCs. Immunohistochemical studies demonstrated the elevation of OPN protein in PDGF-stimulated RASMCs. The temporal expression of OPN mRNA after rat carotid artery balloon angioplasty as assessed by both reverse transcription-polymerase chain reaction and Northern blot analysis revealed a 1.5-fold increase at 6 hours (P < .01) that peaked at 1 and 3 days with a 3.1-fold increase (P < .001). Immunohistochemical studies of carotid artery after angioplasty localized OPN expression in the medical SMCs at 1 day, ie. at a time of significant platelet adherence to the injured vessel, and thereafter to the intimal lesion during neointimal formation. These data suggest that OPN expression in vascular SMCs is regulated by PDGF through the beta-type PDGF receptor in vitro, and possibly in vivo in situations that involve PDGF released from platelets or other cellular sources, such as blood vessels after angioplasty injury.

A Comparative Analysis of Acute-phase Proteins as Inflammatory Biomarkers in Preclinical Toxicology Studies: Implications for Preclinical to Clinical Translation

Recently, in early clinical development, a few biologics and small molecules intended as antitumor or anti-inflammatory agents have caused a severe adverse pro-inflammatory systemic reaction also known as systemic inflammatory response syndrome (SIRS). This toxicity could result from expected pharmacological effects of a therapeutic antibody and/or from interaction with antigens expressed on cells/tissues other than the intended target. Clinical monitoring of SIRS is challenging because of the narrow diagnostic window to institute a successful intervening therapeutic strategy prior to acute circulatory collapse. Furthermore, for these classes of therapeutic agents, studies in animals have low predictive ability to identify potential human hazards. In vitro screens with human cells, though promising, need further development. Therefore, identification of improved preclinical diagnostic markers of SIRS will enable clinicians to select applicable markers for clinical testing and avoid potentially catastrophic events. There is limited preclinical toxicology data describing the interspecies performance of acute-phase proteins because the response time, type, and duration of major acute-phase proteins vary significantly between species. This review will attempt to address this intellectual gap, as well as the use and applicability of acute-phase proteins as preclinical to clinical translational biomarkers of SIRS.

Primary Human Lung Alveolus‐on‐a‐chip Model of Intravascular Thrombosis for Assessment of Therapeutics

Pulmonary thrombosis is a significant cause of patient mortality; however, there are no effective in vitro models of thrombi formation in human lung microvessels that could also assess therapeutics and toxicology of antithrombotic drugs. Here, we show that a microfluidic lung alveolus‐on‐a‐chip lined by human primary alveolar epithelium interfaced with endothelium and cultured under flowing whole blood can be used to perform quantitative analysis of organ‐level contributions to inflammation‐induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet‐endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor‐1 (PAR‐1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development.

Endothelin Receptor Subtype Distribution Predisposes Coronary Arteries to Damage

Several vasoactive drugs that lower blood pressure and increase heart rate induce regional cardiotoxicity in the dog, most frequently of right coronary arteries and right atrium. The basis for this selective damage is thought to result from local changes in vascular tone and blood flow. Administration of an endothelin receptor antagonist (ETRA, SB 209670) to dogs induced damage most frequent and severe in the right coronary artery and right atrium. Because site predisposition may correlate with distribution of vasoactive receptors, the objectives of this study were to map endothelin (ET) receptor distribution and density within regions of dog heart using both gene (mRNA) and protein expression endpoints for dog ET(A) and ET(B) receptors, and, additionally, correlate ET receptor subtype density with regional cardiac blood flow. A 10- to 15-mmHg reduction in mean arterial pressure with a concomitant increase in heart rate (10-20%), a six- and twofold increase in regional blood flow to the right and left atrium, respectively, and acute hemorrhage, medial necrosis, and inflammation were observed in the right coronary arteries and arteries of the right atrium after ETRA infusion for 5 days. Radioligand protein binding to quantify both ET receptors in normal dog heart indicated a twofold greater density of ET receptors in atrial regions versus ventricular regions. Importantly, ET receptor density in coronary arteries was markedly (about five- to sixfold) increased above that in atrial or ventricular tissues. ET receptor subtype characterization indicated ET(B) receptors were three times more prevalent in right coronary arteries compared to left coronary arteries and in situ hybridization confirmed localization of ET(B) in vascular smooth muscle. ET(A) receptor density was comparable in right and left coronary arteries. Quantitative real-time polymerase chain reaction for ET(A) and ET(B) receptor mRNA transcripts supported the site prevalence for message distribution. Consequently, the composite of protein and message expression profiles for ET(A) and ET(B) receptors indicated a disproportionate distribution of ET(B) receptors within right coronary artery of dog and this, along with functional measures of blood flow after ETRA infusion indicated a predisposition for exaggerated pharmacological responses and subsequent damage to right coronary arteries by ET and/or ETRAs.

Time Course of Renal Proximal Tubule Injury, Reversal, and Related Biomarker Changes in Rats Following Cisplatin Administration

Cisplatin (CDDP) is known to produce renal proximal tubule injury. Various renal biomarkers have been related to CDDP nephrotoxicity in previous research, but the temporal and spatial relationship of these biomarkers to injury reversal has not been well defined. In this study, the progression and reversal of renal histopathology findings relative to serum and urinary biomarker changes were examined during a 4-week postdose period following single intraperitoneal administration of CDDP (1 mg/kg) or 0.9% saline. Degeneration, vacuolation, inflammation, and regeneration of the S3 segment of proximal tubules were evident 72 hours following CDDP administration. Tubular degeneration and regeneration were also observed at 1 and 1.5 weeks but at lower incidences and/or severity indicating partial reversal. Complete histologic reversal was observed by 2 weeks following CDDP administration. Urinary kidney injury molecule 1 (KIM-1), α-glutathione-S-transferase (α-GST), and albumin levels increased at 72 hours postdosing, concurrently with the earliest histologic evidence of tubule injury. Changes in urinary KIM-1 correlated with KIM-1 immunostaining in the proximal tubular epithelial cells. No significant changes in serum biomarkers occurred except for a minimal increase in urea nitrogen at 1.5 weeks postdosing. Of the novel renal biomarkers examined, urinary KIM-1, α-GST, and albumin showed excellent concordance with CDDP-induced renal injury progression and reversal; and these biomarkers were more sensitive than traditional serum biomarkers in detecting early, acute renal tubular damage confirmed by histopathology. Furthermore, urinary KIM-1, α-GST, and albumin outperformed other biomarkers in correlating with the time of maximum histologic injury.