Heather M. LaPorte

New Insights into Mechanisms and Functions of Chemokine (C-X-C Motif) Receptor 4 Heteromerization in Vascular Smooth Muscle

Recent evidence suggests that C-X-C chemokine receptor type 4 (CXCR4) heteromerizes with α1A/B-adrenoceptors (AR) and atypical chemokine receptor 3 (ACKR3) and that CXCR4:α1A/B-AR heteromers are important for α1-AR function in vascular smooth muscle cells (VSMC). Structural determinants for CXCR4 heteromerization and functional consequences of CXCR4:α1A/B-AR heteromerization in intact arteries, however, remain unknown. Utilizing proximity ligation assays (PLA) to visualize receptor interactions in VSMC, we show that peptide analogs of transmembrane-domain (TM) 2 and TM4 of CXCR4 selectively reduce PLA signals for CXCR4:α1A-AR and CXCR4:ACKR3 interactions, respectively. While both peptides inhibit CXCL12-induced chemotaxis, only the TM2 peptide inhibits phenylephrine-induced Ca2+-fluxes, contraction of VSMC and reduces efficacy of phenylephrine to constrict isolated arteries. In a Cre-loxP mouse model to delete CXCR4 in VSMC, we observed 60% knockdown of CXCR4. PLA signals for CXCR4:α1A/B-AR and CXCR4:ACKR3 interactions in VSMC, however, remained constant. Our observations point towards TM2/4 of CXCR4 as possible contact sites for heteromerization and suggest that TM-derived peptide analogs permit selective targeting of CXCR4 heteromers. A molecular dynamics simulation of a receptor complex in which the CXCR4 homodimer interacts with α1A-AR via TM2 and with ACKR3 via TM4 is presented. Our findings further imply that CXCR4:α1A-AR heteromers are important for intrinsic α1-AR function in intact arteries and provide initial and unexpected insights into the regulation of CXCR4 heteromerization in VSMC.

Proteasome inhibition prolongs survival during lethal hemorrhagic shock in rats.

BACKGROUND Several lines of evidence suggest that proteasomes, the major nonlysosomal proteases in eukaryotes, are involved in the pathophysiology of various disease processes, including ischemia-reperfusion injury and trauma. Recently, we demonstrated that 26S proteasome activity is negatively regulated by adenosine triphosphate (ATP) and that proteasome activation during ischemia contributes to myocardial injury. The regulation of tissue proteasome activity by ATP and the potential of proteasomes as drug targets during hemorrhagic shock, however, are unknown. Thus, we evaluated the regulation of tissue proteasome peptidase activity and the effects of the proteasome inhibitor bortezomib in rat models of hemorrhagic shock. METHODS Series 1 includes animals (n = 20) hemorrhaged to a mean arterial blood pressure of 30 mm Hg for up to 45 minutes. Series 2 includes animals hemorrhaged to a mean arterial blood pressure of 30 mm Hg for 30 minutes, followed by bortezomib (0.4 mg/kg) or vehicle administration (n =5 per group) and fluid resuscitation until 75 minutes. Series 3 includes animals that underwent 40% blood volume hemorrhage, followed by 2% blood volume hemorrhage every 15 minutes until death. Bortezomib (0.4 mg/kg) or vehicle were administered 15 minutes after the onset of hemorrhage (n = 6–7 per group). Vital signs were continuously monitored. The heart, lung, and pectoral muscle were analyzed for proteasome peptidase activities and levels of ATP, ubiquitin-protein conjugates, and cytokines (tumor necrosis factor &agr;, interleukin 6, and interleukin 10). RESULTS In Series 1, proteasome peptidase activities in tissue extracts increased proportional to the decrease in tissue ATP concentrations during hemorrhagic shock. Activation of proteasome peptidase activity with decreases of the ATP assay concentration was also detectable in normal tissue extracts. In Series 2, systemic administration of bortezomib inhibited tissue proteasome activities but did not affect the physiologic response. In Series 3, bortezomib inhibited tissue proteasome activities, increased endogenous ubiquitin-protein conjugates, and prolonged survival time from treatment from 48.5 minutes in the control group to 85 minutes (p = 0.0012). Bortezomib treatment did not affect tissue cytokine levels. CONCLUSION Proteasome activation contributes to the pathophysiology of severe hemorrhagic shock. Pharmacologic inhibition of the proteasome may provide a survival advantage during lethal hemorrhagic shock.

α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

Background Recently, we provided evidence that α1‐adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C‐X‐C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1‐ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk between ACKR3 and α1‐ARs is unknown. Methods and Results We show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D‐AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D‐AR:ACKR3, CXCR4:ACKR3, and α1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D‐AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D‐AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B‐AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b‐AR in β‐arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine‐induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine‐induced constriction of mesenteric arteries. Conclusions α1‐ARs form hetero‐oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D‐AR function, and activation of ACKR3 negatively regulates α1‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1‐ARs function within a network of hetero‐oligomeric receptor complexes.

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle

Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3–68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05–0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03–3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3–68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3–68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3–68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.

Proteasome Inhibition After Burn Injury

The objective of this study was to assess the effects of proteasome inhibition on the development of burn-induced hypermetabolism. Rats underwent 30–40% total BSA scald burn or sham injury. The proteasome inhibitor bortezomib (0.1 mg/kg) or vehicle (n = 10) was administered i.p. 3× weekly starting at 2 hours (early bortezomib, n = 20) or 48 hours (late-bortezomib, n = 13) postburn. Body weights were determined weekly. Resting energy expenditures (REE) were measured at days 0 (baseline), 7, 14, 21, and 42 postburn. At day 42, blood and pectoral muscle were harvested. Routine blood chemistry parameters were analyzed. Proteasome content, proteasome peptidase activities, and ubiquitin–protein conjugates were measured in muscle extracts. As compared with sham-vehicle-treated animals, specific proteasome activities were increased after burn and vehicle treatment. Bortezomib treatment inhibited proteasome activities and increased ubiquitin–protein conjugates after sham and burn injury. Bortezomib treatment did not affect REE after sham procedure. REE significantly increased by 47% within 7 days and remained elevated until day 42 after burn and vehicle treatment. After early-bortezomib treatment, burn-induced increases in REE were delayed and significantly reduced by 42% at day 42, as compared with vehicle treatment. With late-bortezomib treatment, burn-induced increases in REE were also delayed but not attenuated at day 42. Mortality was 20% with vehicle, 65% (median survival time: 1.875 days) with early-bortezomib and 25% with late-bortezomib treatment after burns (P < .05 early-bortezomib vs vehicle and late-bortezomib). Proteasome inhibition delays development of burn-induced hypermetabolism. Although proteasome inhibition early after burn injury reduces the hypermetabolic response, it significantly increases early burn-associated mortality.

Effects of the Kv7 voltage-activated potassium channel inhibitor linopirdine in rat models of haemorrhagic shock

Recently, we demonstrated that Kv7 voltage‐activated potassium channel inhibitors reduce fluid resuscitation requirements in short‐term rat models of haemorrhagic shock. The aim of the present study was to further delineate the therapeutic potential and side effect profile of the Kv7 channel blocker linopirdine in various rat models of severe haemorrhagic shock over clinically relevant time periods. Intravenous administration of linopirdine, either before (1 or 3 mg/kg) or after (3 mg/kg) a 40% blood volume haemorrhage, did not affect blood pressure and survival in lethal haemorrhage models without fluid resuscitation. A single bolus of linopirdine (3 mg/kg) at the beginning of fluid resuscitation after haemorrhagic shock transiently reduced early fluid requirements in spontaneously breathing animals that were resuscitated for 3.5 hours. When mechanically ventilated rats were resuscitated after haemorrhagic shock with normal saline (NS) or with linopirdine‐supplemented (10, 25 or 50 μg/mL) NS for 4.5 hours, linopirdine significantly and dose‐dependently reduced fluid requirements by 14%, 45% and 55%, respectively. Lung and colon wet/dry weight ratios were reduced with linopirdine (25/50 μg/mL). There was no evidence for toxicity or adverse effects based on measurements of routine laboratory parameters and inflammation markers in plasma and tissue homogenates. Our findings support the concept that linopirdine‐supplementation of resuscitation fluids is a safe and effective approach to reduce fluid requirements and tissue oedema formation during resuscitation from haemorrhagic shock.

Pharmacological modulation of C‐X‐C motif chemokine receptor 4 influences development of acute respiratory distress syndrome after lung ischemia‐reperfusion injury

Activation of C‐X‐C motif chemokine receptor 4 (CXCR4) has been reported to result in lung protective effects in various experimental models. The effects of pharmacological CXCR4 modulation on the development of acute respiratory distress syndrome (ARDS) after lung injury, however, are unknown. Thus, we studied whether blockade and activation of CXCR4 influences development of ARDS in a unilateral lung ischaemia–reperfusion injury rat model. Anaesthetized, mechanically ventilated animals underwent right lung ischaemia (series 1, 30 minutes; series 2, 60 minutes) followed by reperfusion for 300 minutes. In series 1, animals were treated with vehicle or 0.7 μmol/kg of AMD3100 (CXCR4 antagonist) and in series 2 with vehicle, 0.7 or 3.5 μmol/kg ubiquitin (non‐cognate CXCR4 agonist) within 5 minutes of reperfusion. AMD3100 significantly reduced PaO2/FiO2 ratios, converted mild ARDS with vehicle treatment into moderate ARDS (PaO2/FiO2 ratio<200) and increased histological lung injury. Ubiquitin dose‐dependently increased PaO2/FiO2 ratios, converted moderate‐to‐severe into mild‐to‐moderate ARDS and reduced protein content of bronchoalveolar lavage fluid (BALF). Measurements of cytokine levels (TNFα, IL‐6, IL‐10) in lung homogenates and BALF showed that AMD3100 reduced IL‐10 levels in homogenates from post‐ischaemic lungs, whereas ubiquitin dose‐dependently increased IL‐10 levels in BALF from post‐ischaemic lungs. Our findings establish a cause‐effect relationship for the effects of pharmacological CXCR4 modulation on the development of ARDS after lung ischaemia–reperfusion injury. These data further suggest CXCR4 as a new drug target to reduce the incidence and attenuate the severity of ARDS after lung injury.

Ubiquitin Urine Levels in Burn Patients.

The objective of this study was to determine whether urine ubiquitin levels are elevated after burns and to assess whether urine ubiquitin could be useful as a noninvasive biomarker for burn patients. Forty burn patients (%TBSA: 20 ± 22; modified Baux scores: 73 ± 26) were included (control: 11 volunteers). Urine was collected in 2-hour intervals for 72 hours, followed by 12-hour intervals until discharge from the intensive care unit. Ubiquitin concentrations were analyzed by enzyme linked immunosorbent assay and Western blot. Total protein was determined with a Bradford assay. Patient characteristics and clinical parameters were documented. Urine ubiquitin concentrations, renal ubiquitin excretion, and excretion rates were correlated with patient characteristics and outcomes. Initial urine ubiquitin concentrations were 362 ± 575 ng/ml in patients and 14 ± 18 ng/ml in volunteers (P < .01). Renal ubiquitin excretion on day 1 was 292.6 ± 510.8 &mgr;g/24 hr and 21 ± 27 &mgr;g/24 hr in volunteers (P < .01). Initial ubiquitin concentrations correlated with modified Baux scores (r = .46; P = .02). Ubiquitin levels peaked at day 6 postburn, whereas total protein concentrations and serum creatinine levels remained within the normal range. Total renal ubiquitin excretion and excretion rates were higher in patients with %TBSA ≥20 than with %TBSA <20, in patients who developed sepsis/multiple organ failure than in patients without these complications and in nonsurvivors vs survivors. These data suggest that ubiquitin urine levels are significantly increased after burns. Renal ubiquitin excretion and/or excretion rates are associated with %TBSA, sepsis/multiple organ failure, and mortality. Although these findings may explain previous correlations between systemic ubiquitin levels and outcomes after burns, the large variability of ubiquitin urine levels suggests that urine ubiquitin will not be useful as a noninvasive disease biomarker.