Nicholle M. Johnson

Receptor for Advanced Glycation End-Products Regulates Lung Fluid Balance via Protein Kinase C–gp91phox Signaling to Epithelial Sodium Channels

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 μg versus 2.48 ± 0.53 μg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.

An electrically coupled tissue-engineered cardiomyocyte scaffold improves cardiac function in rats with chronic heart failure

BACKGROUND Varying strategies are currently being evaluated to develop tissue-engineered constructs for the treatment of ischemic heart disease. This study examines an angiogenic and biodegradable cardiac construct seeded with neonatal cardiomyocytes for the treatment of chronic heart failure (CHF). METHODS We evaluated a neonatal cardiomyocyte (NCM)-seeded 3-dimensional fibroblast construct (3DFC) in vitro for the presence of functional gap junctions and the potential of the NCM-3DFC to restore left ventricular (LV) function in an in vivo rat model of CHF at 3 weeks after permanent left coronary artery ligation. RESULTS The NCM-3DFC demonstrated extensive cell-to-cell connectivity after dye injection. At 5 days in culture, the patch contracted spontaneously in a rhythmic and directional fashion at 43 ± 3 beats/min, with a mean displacement of 1.3 ± 0.3 mm and contraction velocity of 0.8 ± 0.2 mm/sec. The seeded patch could be electrically paced at nearly physiologic rates (270 ± 30 beats/min) while maintaining coordinated, directional contractions. Three weeks after implantation, the NCM-3DFC improved LV function by increasing (p < 0.05) ejection fraction 26%, cardiac index 33%, dP/dt(+) 25%, dP/dt(-) 23%, and peak developed pressure 30%, while decreasing (p < 0.05) LV end diastolic pressure 38% and the time constant of relaxation (Tau) 16%. At 18 weeks after implantation, the NCM-3DFC improved LV function by increasing (p < 0.05) ejection fraction 54%, mean arterial pressure 20%, dP/dt(+) 16%, dP/dt(-) 34%, and peak developed pressure 39%. CONCLUSIONS This study demonstrates that a multicellular, electromechanically organized cardiomyocyte scaffold, constructed in vitro by seeding NCM onto 3DFC, can improve LV function long-term when implanted in rats with CHF.

Valsartan therapy in heart failure after myocardial infarction: the role of endothelial dependent vasorelaxation.

Angiotensin II receptor blockade (ARB) increases vasorelaxation in heart failure by enhancing endothelial nitric oxide (NO). To determine the effects of valsartan on NO-mediated peripheral vascular function after myocardial infarction (MI), we treated adult male Sprague-Dawley rats immediately after MI with valsartan for 3 weeks (sham, n = 10; MI, n = 11) and 6 weeks (sham, n = 6; MI, n = 8). At both time points, valsartan lowered (P < 0.05) left ventricular (LV) systolic pressure (103 ± 4 and 107 ± 4 vs. 93 ± 3 and 85 ± 4 mm Hg, respectively) and LV end-diastolic pressure (25 ± 1 and 25 ± 2 to 13 ± 2 and 18 ± 3 mm Hg, respectively). Valsartan lowered (P < 0.05) LV dP/dt only at 6 weeks (4676 ± 168 and 4503 ± 232 vs. 4539 ± 281 and 3372 ± 417 mm Hg/sec); valsartan shortened (P < 0.05) the time constant of LV relaxation or tau only at 3 weeks (24.2 ± 1.8 and 26.5 ± 2.3 vs. 20.1 ± 0.7 and 23.8 ± 1.4 msec). At 6 weeks, the vasorelaxation response to acetycholine in aortic rings was decreased (P < 0.05) with MI and improved at acetycholine doses (10−8, 10−7, and 10−4; P < 0.06) with valsartan. Endothelial nitric oxide synthase (eNOS) protein was undetectable in aortic tissue from valsartan treated rats or from aortic tissue incubated with valsartan (2.5, 25, and 50 mg/mL). These data suggest that valsartan improves cardiac function after MI by modulating LV remodeling, decreasing LV end-diastolic pressure, and enhancing both LV diastolic and endothelial function. These effects are mediated, in part, by NO but upregulation of eNOS may not be required for improved systemic endothelial function in heart failure.

Antibody to granulocyte macrophage colony-stimulating factor reduces the number of activated tissue macrophages and improves left ventricular function after myocardial infarction in a rat coronary artery ligation model.

Granulocyte macrophage colony-stimulating factor (GM-CSF) promotes infarct expansion and inappropriate collagen synthesis in a myocardial infarction (MI). This study was designed to determine if treatment with anti-GM-CSF will inhibit macrophage migration, preserve function, and limit left ventricular (LV) remodeling in the rat coronary artery ligation model. Treatment with a monoclonal antibody to GM-CSF (5 mg/kg) was initiated 24 hours before coronary artery ligation and continued every 3 days for 3 weeks. Left coronary arteries of rats were ligated, animals were recovered, and cardiac function was evaluated 3 weeks postligation. Tissue samples were processed for histochemistry. Anti-GM-CSF treatment increased LV ejection fraction (37 ± 3% vs 47 ± 5%) and decreased LV end systolic diameter (0.75 ± 0.12 vs 0.59 ± 0.05 cm) with no changes in LV systolic pressure (109 ± 4 vs 104 ± 5 mm Hg), LV end diastolic pressure (22 ± 4 vs 21 ± 2 mm Hg), LV end diastolic diameter (0.96 ± 0.04 vs 0.92 ± 0.05 cm), or the time constant of LV relaxation tau (25.4 ± +2.4 vs 22.7 ± 1.4 milliseconds) (P < 0.05). Significantly lower numbers of tissue macrophages and significant reductions in infarct size were found in the myocardium of antibody-treated animals (81 ± 21.24 vs 195 ± 31.7 positive cells per 0.105 mm2, compared with controls. These findings suggest that inhibition of macrophage migration may be beneficial in the treatment of heart failure after MI.

Hydrogen Peroxide Stimulates Exosomal Cathepsin B Regulation of the Receptor for Advanced Glycation End-Products (RAGE)

Exosomes are nano‐sized vesicles that are secreted into the extracellular environment. These vesicles contain various biological effector molecules that can regulate intracellular signaling pathways in recipient cells. The aim of this study was to examine a correlation between exosomal cathepsin B activity and the receptor for advanced glycation end‐products (RAGE). Type 1 alveolar epithelial (R3/1) cells were treated with or without hydrogen peroxide and exosomes isolated from the cell conditioned media were characterized by NanoSight analysis. Lipidomic and proteomic analysis showed exosomes released from R3/1 cells exposed to oxidative stress induced by hydrogen peroxide or vehicle differ in their lipid and protein content, respectively. Cathepsin B activity was detected in exosomes isolated from hydrogen peroxide treated cells. The mRNA and protein expression of RAGE increased in cultured R3/1 cells treated with exosomes containing active cathepsin B while depletion of exosomal cathepsin B attenuated RAGE mRNA and protein expression. These results suggest exosomal cathepsin B regulates RAGE in type 1 alveolar cells under conditions of oxidative stress. J. Cell. Biochem. 119: 599–606, 2018.

Angiotensin II regulates δ-ENaC in human umbilical vein endothelial cells

The amiloride-sensitive epithelial sodium channel (ENaC) has been characterized in a variety of non-epithelial tissues. In the current study we sought to understand the effect of angiotensin II on δ ENaC function using human umbilical vein endothelial cells (HUVECs). The δ ENaC subunit is found in humans, but notably absent in rat and most mouse epithelial tissues. In this study we report the presence of δ ENaC in HUVECS with a half-life of ~80min and a change in δ ENaC abundance when HUVECs were treated with angiotensin II. We also observed that angiotensin II increased apical membrane expression of δ ENaC and decreased protein ubiquitination. Equivalent short circuit current measurements showed angiotensin II increased δ ENaC ion transport in HUVEC cells. Treatment with the antioxidant apocynin attenuated angiotensin II mediated effects indicating an important role for angiotensin-derived H2O2 in δ ENaC subunit regulation. Whole cell recordings from oocytes injected with δβγ ENaC shows H2O2-sensitive current. These results suggest that δ ENaC subunits can make up functional channel in HUVEC cells that are regulated by angiotensin II in a redox-sensitive manner. The novel findings have significant implications for our understanding of the role of ENaC in vascular conditions in which oxidative stress occurs.

RAGE-induced changes in the proteome of alveolar epithelial cells

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor and member of the immunoglobulin superfamily. RAGE is constitutively expressed in the distal lung where it co-localizes with the alveolar epithelium; RAGE expression is otherwise minimal or absent, except with disease. This suggests RAGE plays a role in lung physiology and pathology. We used proteomics to identify and characterize the effects of RAGE on rat alveolar epithelial (R3/1) cells. LC-MS/MS identified 177 differentially expressed proteins and the PANTHER Classification System further segregated proteins. Proteins involved in gene transcription (RNA and mRNA splicing, mRNA processing) and transport (protein, intracellular protein) were overrepresented; genes involved in a response to stimulus were underrepresented. Immune system processes and response to stimuli were downregulated with RAGE knockdown. Western blot confirmed RAGE-dependent changes in protein expression for NFκB and NLRP3 that was functionally supported by a reduction in IL-1β and phosphorylated p65. We also assessed RAGEs effect on redox regulation and report that RAGE knockdown attenuated oxidant production, decreased protein oxidation, and increased reduced thiol pools. Collectively the data suggest that RAGE is a critical regulator of epithelial cell response and has implications for our understanding of lung disease, specifically acute lung injury. SIGNIFICANCE STATEMENT In the present study, we undertook the first proteomic evaluation of RAGE-dependent processes in alveolar epithelial cells. The alveolar epithelium is a primary target during acute lung injury, and our data support a role for RAGE in gene transcription, protein transport, and response to stimuli. More over our data suggest that RAGE is a critical driver of redox regulation in the alveolar epithelium. The conclusions of the present work assist to unravel the molecular events that underlie the function of RAGE in alveolar epithelial cells and have implications for our understanding of RAGE signaling during lung injury. Our study was the first proteomic comparison showing the effects of RAGE activation from alveolar epithelial cells that constitutively express RAGE and these results can affect a wide field of lung biology, pulmonary therapeutics, and proteomics.

105 ALTERATIONS IN REGIONAL WALL STRAIN, ENOS AND MICROTUBULIN CONCENTRATIONS SIGNALING LEFT VENTRICULAR REMODELING OCCUR IMMEDIATELY AFTER ACUTE MYOCARDIAL INFARCTION

Background Left ventricular (LV) remodeling after myocardial infarction (MI) leads to heart failure (HF). While alterations in LV wall strain are seen in the infarcted regions (IR), it is unclear if this also affects the non-infarcted regions (NIR) of the LV. Additionally, while LV tissue eNOS and constituitive microtubulin (CM) are altered in chronic HF, it is unknown what happens to these biomarkers acutely. We evaluated changes in LV wall strain; myocardial eNOS and CM acutely post MI. Methods LV wall motion of Sprague Dawley rats (N = 10) was analyzed by echocardiography at several time points after MI. Hemodynamic measurements were obtained via a Millar catheter. Alterations in myocardial contraction (δ strain) are measured via M-mode echocardiography. CM and eNOS were determined via immunoblot techniques. Results Systolic blood pressure decreased (P ≤ 0.05) acutely post MI (127.1 ± 6.1 vs 101.7± 10.1 mmHg). LV end diastolic pressure increased (P≤0.05) acutely after MI (5.8 ± 0.5 vs. 19.9 ± 2.0 mmHg). These changes were accompanied by a decrease (P≤ 0.05) in LV dP/dt after MI (7106 ± 520 vs. 4671± 350, mmHg/sec). δ strain was decreased in the anterior IR immediately (0.15 ± 0.04 vs 0.1 ± 0.02 mm, P ≤ 0.01); similarly δ strain was also decreased in the posterior NIR starting at 1 minute (0.17 ± 0.02 vs 0.1 ± 0.02 mm, P ≤ 0.01). A decrease in eNOS was seen with both the IR and NIR of the LV (20.8± 3.8 intensity unit (IU)/50 μg of tissue vs 11.1 ± 3.3 IU/50 μg, P = 0.04) and (26.7 ± 4.8 vs 8.14 ± 1.6 IU/50 μg, P = 0.002), respectively after MI. Conversely, CM was increased in both IR and NIR of the LV (10.3 ± 2.4 IU/25 μg vs 26.2 ± 4.6 IU/25 μg, P = 0.0005) and (9.3 ± 1.8 vs 18.5 ± 3.7 IU/25 μg, P = 0.003), respectively. Conclusion Decreases in δ strain occurred in both IR and NIR of the LV acutely after MI. This is accompanied by alterations in eNOS and CM levels throughout the LV immediately after MI. Our data demonstrate that physical and biomarkers signaling LV remodeling occur immediately after MI, rather than over time.

Activation of metalloproteinases in systemic conduit arteries after myocardial ischemia

Recent data from our laboratory showed that metalloproteinases (MMPs) and Tissue inhibitor of MMPs (TIMPs) are activated very early after myocardial ischemia. Hemodynamic changes due to myocardial ischemia appear to play a significant role in myocardial MMPs/TIMPs activation. Since the peripheral vasculature is subjected to the same alterations in hemodynamics as the myocardium, we hypothesized that myocardial ischemia and infarction activates peripheral vascular MMPs/TIMPs in a time-dependent manner. MMPs activation was measured in aortic tissue homogenates using gelatin zymography and their abundance was measured using immunoblot analysis. Activation of TIMPs was measured using reverse zymography. MMP activities after ischemia were compared to nonischemic tissue. ProMMP-9 activities were decreased (P 0.05) by 52, 86, 51% at 1 min, 5 min, and 7 days after ischemia, respectively. ProMMP-2 activities were decreased (P 0.05) by 33, 78, 38% at 1 min, 5 min, and 7 days after ischemia, respectively. MMP-2 activities were decreased (P 0.07) by 19% and 17% at 1 min and 5 min after ischemia, respectively. In contrast, MMP-2 activities were increased (P 0.05) by 40% at 7 days after ischemia. ProMMP-9, ProMMP-2 and MMP-2 activities were absent at 21 days after ischemia (Fig.1). Western blot analysis suggests that MMP-2, MMP-9, and MMP-13 may be activated five minutes after ischemia, evident by the disappearance of the proenzymes (high molecular weight bands) and the appearance of the active enzymes (low molecular weight bands) in the ischemic tissue compared to nonischemic vessels (Fig. 2). In addition, TIMP-1 and TIMP-2 activities were decreased (P 0.05) at five minutes, 7 and 21 days after ischemia. Thus, myocardial ischemia modulates the MMPs/TIMPs balance early after ischemia in the peripheral conduit arteries.