P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1.

The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression correlates with end-organ protection regardless of overt antihypertensive action.

Differential alterations in 5alpha-reductase type 1 and type 2 levels during development and progression of prostate cancer.

BACKGROUND: In the prostate, conversion of testosterone to dihydrotestosterone (DHT), by the enzymes 5alpha-reductase types 1 and 2 (5alphaR1, 5alphaR2) is required for normal growth and probably also for development of prostate cancer (PCa). Finasteride, a 5alphaR2 inhibitor, was shown to reduce the prevalence of PCa in the Prostate Cancer Prevention Trial. However, inhibition of both 5alphaR isoenzymes causes a greater decrease in serum DHT. The aim of this study was to assess differential expression of these enzymes at various stages of PCa development. METHODS: Immunostaining for 5alphaR1 and 5alphaR2, using specific, well-validated antibodies, was evaluated in 26 benign prostatic hyperplasia (BPH) (16 for 5alphaR2), 53 primary PCa (21 for 5alphaR2), 18 prostatic intraepithelial neoplasia (PIN), 12 primary PCa treated with neoadjuvant androgen ablation, 15 locally recurrent PCa specimens, and 18 PCa metastases. RESULTS: The mean area of moderate plus high intensity staining for 5alphaR1 increased from 4.8 +/- 2.8% of total epithelial area in BPH, to 18.9 +/- 5.7% in PIN, 17.0 +/- 3.2% in primary cancer, 38.0 +/- 7.3% in recurrent cancer, and 55.8 +/- 8.5% in PCa metastases. The mean staining area for 5alphaR2 decreased from 58.8 +/- 7.2% in BPH, to 21.1 +/- 5.5% in PIN and 34.8 +/- 6.7% in primary PCa. Staining for 5alphaR2 was increased in recurrent cancer and PCa metastases compared to primary PCa, at 58.7 +/- 5.2% and 69.2 +/- 8.7%, respectively. CONCLUSIONS: 5alphaR1 immunostaining is increased and 5alphaR2 immunostaining is decreased during development of PCa. In addition, there is increased expression of both 5alphaR isozymes in recurrent and metastatic cancers, suggesting that both isozymes may be important in the development and progression of PCa.

Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization.

Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis. This actin remodeling is regulated in part by Rho family GTPases. Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases. Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development. Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance. Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies. Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II. Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies. Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin. Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events. Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B. We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.

Clusterin expression in adult human normal and osteoarthritic articular cartilage.

OBJECTIVE: To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage. METHODS: Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization. RESULTS: Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. CONCLUSION: The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.

Skeletal muscle deformity and neuronal disorder in Trio exchange factor-deficient mouse embryos.

Dbl-homology guanine nucleotide exchange factors (DH-GEFs) regulate actin cytoskeletal reorganization, cell adhesion, and gene transcription via activation of Rho GTPases. However, little is known about the physiological role of mammalian DH-GEFs during development. The DH-GEF family member Trio is of particular interest because it is a multifunctional protein possessing two GEF domains, as well as a protein serine/threonine kinase domain, and trio-like genes in Caenorhabditis elegans and Drosophila were shown to function in neural migration and axon guidance. To determine the role of Trio during mammalian development, we generated a mouse trio loss-of-function mutation (trio(-/-)). Trio function is essential during late embryonic development as genotype analysis indicated that trio(-/-) embryos died between embryonic day (E)-15.5 and birth, or shortly thereafter. In the trio(-/-) embryos, primary skeletal myofibers were relatively normal at E14.5, but by E18.5 highly unusual spherical myofibers accumulated. Trio deficiency may cause a defect in secondary myogenesis, as the appearance of the abnormal trio(-/-) skeletal myofibers temporally coincided with the onset of secondary myogenesis, and smaller secondary myofibers located adjacent to the primary myofibers were absent. The proliferation of trio(-/-) secondary myoblasts appeared normal, suggesting that Trio may regulate secondary myoblast alignment or fusion. trio(-/-) embryos also displayed aberrant organization in several regions within the brain, including the hippocampal formation and olfactory bulb. We thus conclude that Trio is essential for late embryonic development, and that Trio functions in fetal skeletal muscle formation and in the organization of neural tissues.

The trio guanine nucleotide exchange factor is a RhoA target. Binding of RhoA to the trio immunoglobulin-like domain.

Trio is a complex protein containing two guanine nucleotide exchange factor domains each with associated pleckstrin homology domains, a serine/threonine kinase domain, two SH3 domains, an immunoglobulin-like domain, and spectrin-like repeats. Trio was originally identified as a LAR tyrosine phosphatase-binding protein and is involved in actin remodeling, cell migration, and cell growth. Herein we provide evidence that Trio not only activates RhoA but is also a RhoA target. The RhoA-binding site was mapped to the Trio immunoglobulin-like domain. RhoA isoprenylation is necessary for the RhoA-Trio interaction, because mutation of the RhoA carboxyl-terminal cysteine residue blocked binding. The existence of an intramolecular functional link between RhoA activation and RhoA binding is suggested by the finding that Trio exchange activity enhanced RhoA binding to Trio. Furthermore, immunofluorescence studies of HeLa cells showed that although ectopically expressed Trio was evenly distributed within the cell, co-expression of Trio with RhoA resulted in relocalization of Trio into punctate structures. Relocalization was not observed with Trio constructs lacking the immunoglobulin-like domain, indicating that RhoA acts to regulate Trio localization via binding to the immunoglobulin-like domain. We propose that Trio-mediated RhoA activation and subsequent RhoA-mediated relocalization of Trio functions to modulate and coordinate Trio signaling.

Complications of lower extremity arteriovenous grafts in patients with end-stage renal disease.

BACKGROUND: More data are needed to assess lower extremity angioaccess sites for hemodialysis. METHODS: We did a retrospective review of 843 consecutive hospital records of upper and lower extremity arteriovenous (AV) fistulas from 1992 to 1996. RESULTS: Lower extremity grafts accounted for 16% (134/843) of patients in this series. Complications occurred in 58 of 134 patients (43%) and were more prevalent in women, blacks, diabetic, and hypertensive patients, but not of statistical significance. Dialysis was done for a mean duration of 13.3 years, with a mean graft patency rate of 13.8 months. The 12-month survival rate of lower extremity AV grafts was 62% (83/134). Complications in the lower extremity AV graft group (58 patients) included infections in 27 patients (46%), thrombosis within 30 days in 16 (28%), pseudoaneurysm in 9 (16%), and graft hemorrhage in 6 (10%). CONCLUSIONS: There is a decreased patency rate in lower extremity AV grafts.

A modified metal-ion affinity chromatography procedure for the purification of histidine-tagged recombinant proteins expressed in Drosophila S2 cells.

We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni-NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with the binding of His-tagged protein to the charged resin. In contrast, this modified IMAC procedure, using chelating Sepharose instead of Ni-NTA, enables efficient purification from copper-containing medium in a single step. This method appears to rely upon a preferential interaction of protein-copper complexes with immobilized chelating resin. We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpressing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method should be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free metal ions are present.

Trio amino-terminal guanine nucleotide exchange factor domain expression promotes actin cytoskeleton reorganization, cell migration and anchorage-independent cell growth.

Rho family GTPases regulate diverse cellular processes, including extracellular signal-mediated actin cytoskeleton reorganization and cell growth. The functions of GTPases are positively regulated by guanine nucleotide exchange factors, which promote the exchange of GDP for GTP. Trio is a complex protein possessing two guanine nucleotide exchange factor domains, each with adjacent pleckstrin homology and SH3 domains, a protein serine/threonine kinase domain with an adjacent immunoglobulin-like domain and multiple spectrin-like domains. To assess the functional role of the two Trio guanine nucleotide exchange factor domains, NIH 3T3 cell lines stably expressing the individual guanine nucleotide exchange factor domains were established and characterized. Expression of the amino-terminal guanine nucleotide exchange factor domain results in prominent membrane ruffling, whereas cells expressing the carboxy-terminal guanine nucleotide exchange factor domain have lamellae that terminate in miniruffles. Moreover, cells expressing the amino-terminal guanine nucleotide exchange factor domain display more rapid cell spreading, haptotactic cell migration and anchorage-independent growth, suggesting that Trio regulates both cell motility and cell growth. Expression of full-length Trio in COS cells also alters actin cytoskeleton organization, as well as the distribution of focal contact sites. These findings support a role for Trio as a multifunctional protein that integrates and amplifies signals involved in coordinating actin remodeling, which is necessary for cell migration and growth.

What is the paradigm: hospital or home health care for pressure ulcers?

A home health care (HHC) referral should link the patient in a cost-effective fashion to the physician, home care, and instructions regarding ulcer management. Twenty-one patients (mean age, 74.6 years) had stage III pressure ulcers (<100 cm2) and an involved family member at home. Risk and contributing factors included cardiac disease (n = 9), hypertension (n = 14), end-stage renal disease (n = 7), smoking (n = 11), diabetes (n = 8), chronic brain syndrome (n = 14), cerebrovascular accident (n = 5), and above-the-knee amputation (n = 2). Treatment regimens included standard wound care, pressure relief and, where appropriate, culture-specific antibiotics, as well as a rehabilitation program. Home care progressively decreased the frequency of the nurse HHC and physician office visits. Resolution of the pressure ulcer varied from 6 to 32 weeks. Only two patients had progression of their wound and required hospital readmission. The billable fees included: 1) an office visit, $30.00 (medicare reimbursement, $14.00); 2) the HHC nurse visit, $159.00 (medicare reimbursement, $105.00); 3) supplies, $75.00 to $150.00/week (variable reimbursement); 4) hospitalization, $400.00 to $900.00/day; and 5) a chronic-care bed, $400.00 to $750.00/day. HHC, given a responsible support team and an involved family member, was more socially and financially acceptable than an inpatient facility. Intermittent physician visits with HHC proved safe and reliable, with 90 per cent successfully healing their wounds.

Epidemiology, risk factors, and management of peripheral vascular disease.

Peripheral vascular occlusive disease is initiated with a genetic risk factor component compounded by patient-controlled contributions including obesity, diabetes, hypertension, and smoking. Medical management of these factors may delay or obviate surgical intervention. Angiography may be used to perform angioplasty (+/- stents) or to guide various interventional procedures. The major contribution to pre- and post-operative assessment is the noninvasive laboratory.

Erythropoietin therapy improves graft patency with no increased incidence of thrombosis or thrombophlebitis.

BACKGROUND: Recombinant human erythropoietin (rHuEPO) for the treatment of severe anemia in patients with end-stage renal disease (ESRD) is suggested to improve rehabilitation and cognitive function. The criticism is the alleged increase in the failure rate of arteriovenous (AV) access grafts and in the incidence of lower-extremity deep venous thrombophlebitis (DVT). This study addressed the longevity of AV grafts and the incidence of DVT. STUDY DESIGN: We reviewed 481 consecutive patients with ESRD on dialysis with PTFE access grafts, including 173 consecutive patients who were receiving rHuEPO and 308 who were not. rHuEPO was administered during dialysis titrated against the hematocrit to achieve a level of 33% to 38%. The rHuEPO-ESRD group included 173 patients with a mean age of 58 years, including 54% women; 84% of the grafts were in the upper extremity. In the control group of 308 patients, 57% were women. Diabetes and hypertension were controlled in both groups. RESULTS: Forty-five of 173 rHuEPO patients (26%) experienced graft thrombosis within 1 year. Among 88 episodes of thrombosis, 14 patients experienced multiple episodes. Primary patency was 8.9 months; secondary patency was 11.2 months. In the control population, 95 of 308 patients (31%) experienced graft thrombosis; 27 patients had multiple episodes. Primary patency was 7.8 months and secondary patencywas 9.8 months. The hematocrit improved from a mean of 23% in the control group to 34% in the treated rHuEPO group. Two patients in the control group and one patient receiving rHuEPO experienced DVT in the lower extremity. CONCLUSIONS: Primary and secondary AV fistula patency rates were improved by 10% with rHuEPO. rHuEPO did not increase DVT.

Peripheral neuropathies in diabetes.

Peripheral neuropathy in diabetes remains a difficult management dilemma. The clinical manifestations may vary widely. Polyneuropathies develop with increasing duration of disease, and a thorough understanding of the clinical manifestations, including sensory, motor, and autonomic deficiencies, helps guide diagnosis and treatment. A multidisciplinary approach emphasizing preventive care and timely intervention can decrease morbidity significantly and improve the quality of life for the patient. Properly fitting shoes and avoidance of foot trauma are cornerstones of preventive management. Strict control of serum glucose can alter the course of peripheral neuropathies. This control can be accomplished with a strict insulin regimen or pancreatic transplant. Further research is needed to increase knowledge about peripheral neuropathies in diabetes and aid the physician with new treatment options.

Sequential biannual prevalence studies of pressure ulcers at Allegheny-Hahnemann University Hospital.

To assess the prevalence, documentation and care of pressure ulcers, and the effect of teaching and prevention strategies in a 750-bed university hospital, one-day studies were conducted in 1993, 1995, and 1997. Data gathered was used to evaluate areas in need of improvement and find cost-effective ways to reduce the prevalence of pressure ulcers. The overall prevalence of ulcers decreased from 18 percent in 1993 to 10 percent in 1995 and 1997. The prevalence of nosocomial ulcers decreased from 14 percent in 1993 to 8 percent in 1995 and 6 percent in 1997. The number of nutritional consults increased from 54 percent in 1993 to 67 percent in 1997, and more than half of all patients tested had serum albumin levels < 3.5 mg/dL. Skin assessments upon admission were completed in the majority of patients. While ulcer documentation was less than adequate for the majority of patients in 1993 and 1997, care measures, e.g., placement of patients on specialty beds or mattresses and use of dressings that provide a moist environment, improved considerably. The results of this study indicate that system-wide educational efforts aimed at all levels of patient care providers, and multi-specialty prevention and care efforts can reduce the prevalence of pressure ulcers.

Axillobifemoral bypass: elective versus emergent operation.

Axillobifemoral bypass (AxBFB) is considered an inferior operation because of comparatively poor long-term results. One factor that has not been considered in the literature is whether or not the operation is performed electively or for acute ischemia (< 24 hours duration). This may be a more important predictor of poor results than previously recognized. During the last 10 years, we have performed 59 AxBFB. In Group A, 41 patients (mean age 71) underwent elective AxBFB and in Group B, 18 patients (mean age 65) had emergency AxBFB. Indications for surgery in Group A were limb-threatening ischemia (30), infected aortic graft (5), and severe claudication (6); in Group B, indications for surgery were acute limb ischemia (16), and aortoduodenal fistula (2). Primary patency (p < 0.002), limb salvage (p < 0.002), and survival (p < 0.03) were significantly better in Group A versus Group B. We conclude that an AxBFB performed electively provides satisfactory palliation of severe vascular disease in high-risk patients. The indications for operation and timing of the operation may explain the widely disparate clinical results reported in the literature.

Determination of carotid plaque risk by ultrasonic tissue characterization.

This in vitro study investigated the ability of ultrasonic tissue characterization (UTC) to discriminate between plaques from asymptomatic and symptomatic patients and to compare UTC findings with quantitative measurements of plaque morphology. A total of 34 plaque specimens removed at carotid endarterectomy were scanned transversely at intervals of 1 mm, and compared to tissue cross-sections examined by optical microscopy employing computer-assisted planimetry. UTC was performed by spectral analysis of backscattered radiofrequency signals. The slope, intercept and total power parameters of the spectrum were evaluated. Discriminant analysis was used to compare the ability of the UTC spectral parameters and morphological constituents to correctly classify plaques according to their symptom group membership. UTC correctly classified 88.2% of the plaques. Thrombus was present in 93.9% of the plaques, and there was little difference in the morphological constituents of plaques from asymptomatic and symptomatic patients. Morphological constituents correctly classified 60.7% of the plaques. We conclude, in this preliminary study, that UTC can discriminate between carotid plaques from asymptomatic and symptomatic patients with moderate accuracy, despite a similarity in their morphological composition. UTC discrimination is not related to differences in the type or amount of morphological constituents in the plaques.

Individuality and relatedness of the self: an autophotographic study.

Using R. C. Ziller's (1990) method of autophotography, the authors studied the duality of relatedness and individuality. A total of 226 young adults used photos (and words) to portray "who they are". Photo essays were scored for objective codes (e.g., people touching) and subjectively rated for individuality (richness of self-depiction). As predicted, persons who emphasized their personal rather than social identity depicted greater individuality; relatedness was associated with social and collective identity. Other results replicated earlier gender differences in relatedness and associations of individuality and relatedness with the 5-factor model. In the 2nd wave of the sample, individuality was associated with higher ego development, maturity, and self-directed values; low individuality was associated with restrictive conformity and security values. The authors suggest that the combination of individuality and relatedness is optimal for human development.