Alan R. Parrish

Renal ischemia reperfusion inhibits VEGF expression and induces ADAMTS-1, a novel VEGF inhibitor

Reductions in vascular density occur following acute ischemia-reperfusion (I/R) injury that may predispose the development of chronic kidney disease. The mechanisms mediating vascular loss are not clear but may relate to the lack of effective vascular repair responses. To determine the regulation of the VEGF/VEGFR pathway following I/R injury, male Sprague-Dawley rats were subjected to bilateral renal ischemia (45 min) and allowed to recover for 1, 3, 7, and 35 days. VEGF mRNA expression was repressed by greater than 50% of control values up to 3 days postischemia, while VEGF protein was repressed for up to 7 days postischemia. The renal mRNA expression of receptors was not altered postischemia; however, VEGFR1 (flt-1) protein was transiently reduced in kidney while soluble flt-1 was elevated in plasma at 7 days following injury. Microarray analysis of angiogenesis-related genes identified the enhanced expression of a number of genes, among these was ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif-1), a secreted VEGF inhibitor. The altered expression of ADAMTS-1 was confirmed using RT-PCR and Western blot analysis; immunofluorescence localized its expression to proximal tubules following I/R injury. Other genes identified using microarray included aminopeptidase N, Smad-1, and Id-3 and their localization was also examined using immunohistochemistry. In summary, the data indicate no clear pattern of anti-angiogenic gene expression following renal I/R injury. However, the studies do suggest an overall inhibition of the VEGF pathway during the early injury and repair phase of renal ischemia that may contribute to an overall reduction in renal microvascular density.

Atypical cytochrome P450 induction profiles in glomerular mesangial cells at the mRNA and enzyme level: Evidence for CYP1A1 and CYP1B1 expression and their involvement in benzo[a]pyrene metabolism

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P4501B1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 microM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1A1 mRNA levels, a response abolished by cotreatment with 10 microM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with alpha-naphthoflavone (alpha NF, 1 microM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 microM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 microM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with alpha NF (1 microM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.

Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology

Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCl2, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl2 demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology.

Loss of N-cadherin and α-catenin in the proximal tubules of aging male Fischer 344 rats

Abstract Aging is associated with a loss of renal reserve, and increased sensitivity to either xenobiotic or physiologic insult. Given the critical role of the cadherin/catenin complex in establishing and maintaining the integrity and polarity of tubular epithelial cells, it was hypothesized that aging was associated with alterations in renal cadherin/catenin complexes. Histological assessment of aged (24 months) kidneys harvested from male Fischer 344 rats demonstrates mild degeneration of proximal tubules, multifocal chronic lymphocytic infiltration, moderate development of protein casts inside tubules, and tubular dilatation or degeneration. Western blot analysis revealed that N-cadherin protein expression is not constant over 24 months. N-cadherin expression increased from 4 to 9 months, with peak levels at 9 and 13 months. A decrease in expression was seen at 19 months and an almost complete loss of expression was seen at 24 months. In contrast, the expression of E- and Ksp-cadherin was constant over 24 months. A loss of α-catenin at was seen at 19 and 24 months in the absence of changes in β-, γ-, and p120-catenin. This pattern of N-cadherin expression (increase followed by decrease) was confirmed by real-time PCR analysis, which demonstrated a similar pattern as the Western blot, suggesting that the loss of N-cadherin protein was due to decreased gene expression. The loss of N-cadherin was specific for the kidney, as no changes in N-cadherin expression in the liver, brain, or testes were seen during aging. The conclusion that loss of N-cadherin expression is a critical component of the renal dysfunction associated with aging is supported by the finding that caloric restriction attenuates the loss of N-cadherin, as well as the finding that a significant loss of N-cadherin is seen in the kidneys of ZDF×SHHF rats, a genetic model of end-stage renal disease. Cadherin and catenin expression was further analyzed by immunofluorescence. A significant loss of staining of both N-cadherin and α-catenin was seen in the proximal tubules of rats at 24 months. Interestingly, this corresponded with delocalization of the α-1 subunit of the Na+K+-ATPase, i.e. aberrant staining on cell–cell borders and some indication of apical staining in proximal tubules. Taken together, these data suggest that aging is associated with decreased expression of N-cadherin and α-catenin and is associated with a loss of cell polarity.

Effects of early postnatal ethanol intubation on GABAergic synaptic proteins

Fetal alcohol syndrome includes brain damage from aberrant synaptogenesis, altered cell-cell signaling and blunted plasticity in surviving neurons. Distortion of neurotrophic GABA signals by ethanol-mediated allosteric modulation of GABA(A) receptor (GABA(A)R) activity during brain maturation may play a role. In this regard, early postnatal binge-like ethanol treatment on postnatal days (PDs) 4-9 acutely inhibits whole cell GABA(A)R Cl(-) current and subsequently blunts GABA(A)R function in medial septum/diagonal band (MS/DB) neurons and cerebellar Purkinje cells [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. In light of these functional changes, we hypothesized that ethanol treatment also would decrease levels of proteins important for assembly of GABAergic synapses in maturing brain. To test this relationship, binge-like ethanol intubation was administered to rat pups on PDs 4-9 producing peak blood ethanol concentrations in the range of 302.5+/-6.3 mg/dl. GABAergic synaptic proteins were measured in brain tissue on PDs 13-14 when GABA(A)R currents in individual MS/DB neurons are reduced, but those of cerebellar Purkinje neurons are not yet altered [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. Surprisingly, ethanol did not decrease protein levels of GABA(A)R alpha1/beta2 subunits, GAD(67) or gephyrin in MS/DB at this time when whole cell recordings indicate GABA(A)R function is impaired in acutely dissociated individual neurons. However, in cerebellum where ethanol treated Purkinje cell GABA(A)R function remains normal on PDs 13-14 [Brain Res. 832 (1999) 124-135], reduced levels of several GABAergic synaptic proteins including: GAD(67), GABA(A)R alpha1 subunit, ClC-2 a voltage-gated Cl(-) channel, synaptotagmin a synaptic vesicle protein, and N-cadherin, a synapse associated cell adhesion molecule, were found. These results indicate that binge-like ethanol exposure differentially decreases GABAergic synaptic proteins in some brain areas in a pattern that does not parallel reductions in GABA(A)R function of individual neurons that survive this ethanol insult.

Developing Disaster Preparedness Competence: An Experiential Learning Exercise for Multiprofessional Education

Background: The hurricane disasters of 2005 and the threat of pandemic infectious diseases compel medical educators to develop emergency preparedness training for medical students and other health care professional students. Description: This article describes an experiential exercise for learning a number of the general core competencies in the 2003 AAMC report titled “Training Future Physicians about Weapons of Mass Destruction.” A modified tabletop exercise for medical and veterinary students, which was developed and implemented in 2005, is described. The exercise focused on Highly Pathogenic Avian Influenza (HPAI), an emerging infectious disease scenario that raised the possibility of biological attack. The students were assigned roles in small groups, such as community physicians, hospital personnel, public health officials, veterinarians, school nurses, and emergency managers. Fifteen faculty members were recruited from these various areas of expertise. Pre- and posttesting of medical students showed significant gains in knowledge. The authors describe the scenario, small-group role playing, study questions, injects, Web sites and readings, and evaluation tools. Conclusions: This experiential exercise is an effective, inexpensive, and easily adapted tool for promoting multiple competencies in mass health emergency preparedness for a variety of health care students including medical, veterinary, public health, and nursing students.

Triazine Dendrimers for Drug Delivery: Evaluation of Solubilization Properties, Activity in Cell Culture, and In Vivo Toxicity of a Candidate Vehicle

Three criteria are evaluated to assess the potential of a dendrimer based on triazines, 1, for use as a vehicle for drug delivery. These criteria are: (1) its ability to solubilize small hydrophobic guests as measured spectrophotometrically; (2) its ability to deliver a drug in vitro as evaluated using a gene reporter assay; and (3) its in vivo toxicity in mice as determined by autopsy and screens of liver and kidney function. Vehicle 1 solubilizes pyrene to a similar extent to dendrimers based on poly(arylether)s, 4, encapsulating approximately 0.2 molecules of pyrene per dendrimer. This activity is approximately 10-fold greater than that of the more polar poly(propyleneimine) and poly(amidoamine) dendrimers, 2 and 3. Gas-phase computational models reveal that both 1 and 4 have cores that are accessible to solvent, suggesting that these dendrimers can occupy much greater volumes than 2 and 3 whose cores are confined toward the interior of the structure. Electrostatic potential maps can be used to rationalize differences in solubilization between 1 and 4. Precipitation results from mixing cationic 1 with the anionic indomethacin, but not with methotrexate, suggesting that the composition of the drug may dictate the scope of delivery applications. Dendrimer 1 solubilizes 10-hydroxycamptothecin and a novel bisindolemethane; approximately four and five molecules of drug per dendrimer are solubilized, respectively. In cell-culture experiments using a luciferase reporter gene assay, the dendrimer:bisindolemethane conjugate shows comparable activity to the bisindolemethane delivered in aqueous DMSO, suggesting that the dendrimer does not preclude delivery of the molecule to an intracellular target. Preliminary toxicology studies of 1 in mice show that this molecule has no adverse toxicity to the kidneys or the liver in single doses delivered intraperitoneally up to 10 mg/kg.

Growth-related signaling as a target of toxic insult in vascular smooth muscle cells: Implications in atherogenesis

Aberrant smooth muscle cell proliferation is a focal point in the genesis and progression of atherosclerosis. To date, limited information is available on the molecular and cellular basis of the atherogenic response and the potential contribution of environmental chemicals to the overall process. This review highlights major findings in this laboratory on the mechanism(s) responsible for the acquisition of a proliferative phenotype in vascular smooth muscle cells following repeated cycles of treatment with allylamine and benzo(a)pyrene, known atherogenic chemicals. These agents share the ability to induce and promote aberrant proliferative behavior in smooth muscle cells, but appear to interfere with distinct molecular targets.

Single-cell RT-PCR detects shifts in mRNA expression profiles of basal forebrain neurons during aging

The medial septum and nucleus of the diagonal band (MS/nDB) contain cholinergic and GABAergic neuronal populations that have been identified based on immunohistochemical staining and/or electrophysiological properties. We explored the molecular diversity of MS/nDB neurons using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) to assess gene expression profiles during aging in individual neurons acutely isolated from young (2-4 months) and aged (26-27 months) F344 rats. Neuronal gene expression profiles were characterized by detection of mRNAs for choline acetyltransferase (ChAT, cholinergic) and glutamate decarboxylase (GAD67, GABAergic), as well as mRNAs for calcium binding proteins (CaBPs) calbindin-D28k, calretinin and parvalbumin. Four major neuronal populations were identified: ChAT-positive (ChAT+) cells, GAD-positive (GAD+) cells, ChAT+/GAD+ cells and ChAT negative/GAD negative (ChAT-/GAD-) cells. With age, the percentage of cells expressing ChAT mRNA decreased from 53% in young to 40%, and the expression of GAD67 mRNA was reduced from 56 to 35% of the cells tested. The percentage of cells with detectable levels of both ChAT and GAD67 mRNA was reduced from 24% in young to 9% in aged. Concomitantly, the percentage of ChAT-/GAD- cells increased from 15 to 34% with age. Of the CaBPs, calretinin expression was observed most frequently in this study, and its detection decreased from 33 to 22% of the cells with age. Observations concerning the CaBPs were confirmed using in situ hybridization. These results suggest a shift in the mRNA expression profiles of MS/nDB neuronal populations during aging and exemplify the molecular diversity of cholinergic and GABAergic cells.

Differential processing of osteopontin characterizes the proliferative vascular smooth muscle cell phenotype induced by allylamine

Repeated cycles of vascular injury by allylamine induce vascular lesions similar to those seen in atherosclerotic vessels, or following balloon catheterization. Vascular (aortic) smooth muscle cells harvested from allylamine‐treated animals (i.e., allylamine cells) acquire a proliferative advantage relative to control counterparts that is associated with differential secretion and extracellular matrix sequestration of several proteins. In the present study, we have characterized two of these proteins (Mr 52 and 36 kDa; pl 5.6 and 5.2, respectively) and their putative role in the expression of a proliferative phenotype. Because the physical properties of these proteins were comparable to those of osteopontin (OPN) and its thrombin‐generated fragment(s), initial experiments were conducted to examine the expression and processing of OPN in this cell system. OPN mRNA expression was enhanced during early G1 cell cycle progression in allylamine cells relative to control counterparts. However, comparable amounts of OPN (Mr 56, 52, and 50 kDa) were detected by Western analysis in media conditioned by both cell types using the OP‐199 or B77‐Rat1 antibodies to OPN. Allylamine cells, however, produced increased amounts of a 36 kDa protein recognized by the OP‐199 antibody. Incubation of conditioned media from [35S]methionine‐labeled allylamine cells with thrombin decreased the intensity of the 52 kDa protein, while increasing the intensity of a 36 kDa protein. RT‐PCR analysis demonstrated expression of a 1.2 kb OPN band in both cell types consistent with the predicted size of OPN mRNA, suggesting that the 36 kDa fragment recognized by OP‐199 in allylamine cells was likely not due to altered splicing of the OPN transcript. To determine if OPN and/or the 36 kDa fragment played a central role in the proliferative capacity of allylamine cells, the effect of an antibody to an αv integin subunit was examined. An antibody to the αv subunit, but not α4, nullified the proliferative advantage of allylamine cells relative to control counterparts, suggesting that integrin‐mediated signaling is a key feature of the proliferative phenotype of allylamine cells. We conclude that enhanced proteolytic cleavage of OPN may characterize the modulation of vascular SMCs to a more proliferative phenotype following chemical injury by allylamine. J. Cell. Biochem. 65:267–275.