Heparanase in Acute Kidney Injury.

Recent years have brought about fledgling realization of the role played by heparanase in the pathogenesis of diverse diseases including kidney diseases and, specifically, acute kidney injury. Human heparanase-1 is critically and uniquely engaged in cleavage of heparan sulfate, an integral part of glycocalyx and extracellular matrix where it harbors distinct growth factors, cytokines, and other biologically active molecules. The enzyme is induced and activated in acute kidney injury regardless of its causes, ischemic, nephrotoxic, septic or transplantation-related. This event unleashes a host of sequelae characteristic of the pathogenesis of acute kidney injury, such as induction and reinforcement of innate immune responses, predisposition to thrombosis, activation of monocytes/macrophages and remodeling of the extracellular matrix, thus setting up the stage for future fibrotic complications and development of chronic kidney disease. We briefly discuss the emerging therapeutic strategies of inhibiting heparanase, as well as the diagnostic value of detecting products of heparanase activity for prognostication and treatment.

Therapeutic Restoration of Endothelial Glycocalyx in Sepsis.

Endothelial glycocalyx (EG) is disintegrated during sepsis. We have previously shown that this occurs very early in the course of sepsis and its prevention improves the survival of mice with sepsis. Here, we sought to investigate the possibility of pharmacologically accelerating the restoration of disintegrated EG in sepsis. We used a soilage injection model to induce polymicrobial sepsis in C57/BL6 mice and measured total body EG. En face aortic preparations were used for staining of markers of EG and atomic force microscopy was used to measure EG in vitro. In vitro studies were conducted in cultured endothelial cells either exposed to a lipopolysaccharide or enzymatically denuded of EG. Sulodexide (SDX), a heparin sulfate-like compound resistant to degradation by heparanase, accelerated EG regeneration in vitro and in vivo. The total volume of EG was drastically reduced in septic mice. Administration of SDX produced a dramatic acceleration of EG restoration. This effect, unrelated to any SDX-induced differences in microbial burden, was associated with better control of vascular permeability. Notably, SDX demonstrated not only a remarkable capacity for EG regeneration in vitro and in vivo but was also associated with improved animal survival, even when instituted 2 hours after induction of severe sepsis. In conclusion, 1) EG is disintegrated in sepsis, the event which contributes to high animal mortality; 2) pharmacologic acceleration of EG restoration can be achieved using SDX; and 3) SDX reduces vascular permeability, which is elevated in septic mice, and improves animal survival.

Stress-Induced Premature Senescence of Endothelial and Endothelial Progenitor Cells.

This brief overview of premature senescence of dysfunctional endothelial and endothelial progenitor cells provides information on endothelial cell differentiation and specialization, their ontogeny, and controversies related to endothelial stem and progenitor cells. Stressors responsible for the dysfunction of endothelial and endothelial progenitor cells, as well as cellular mechanisms and consequences of endothelial cell dysfunction are presented. Metabolic signatures of dysfunctional endothelial cells and senescence pathways are described. Emerging strategies to rejuvenate endothelial and endothelial progenitor cells conclude the review.

Anemia, inflammation and health-related quality of life in chronic kidney disease patients.

BACKGROUND: There is controversy regarding whether an incremental increase in hemoglobin levels is associated with improvements in health-related quality of life (HRQOL) in chronic kidney disease (CKD) patients treated with erythropoiesis-stimulating agents (ESAs). We hypothesized that HRQOL in anemic CKD patients has a multifactorial etiology, including the effects of anemia and inflammation. METHODS: 69 non-dialysis CKD patients over 18 years of age with a mean estimated glomerular filtration rate (eGFR) of 43.7 ± 28.8 ml/min/1.73 m2 were divided into anemic and non-anemic cohorts. Kidney disease quality of life (KDQOL) was prospectively recorded using Short Form (SF)-36 components of KDQOL-SF-™ version 1.3 questionnaire. Inflammation was assessed by using a composite of interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α levels in the upper two quartiles. RESULTS: Anemic patients had significantly worse SF-36 components of KDQOL-SF-™ version 1.3, including SF-12 mental component (p = 0.02), role emotional (p = 0.002) and physical function (p = 0.01) compared to patients without anemia. However, in multiple linear regression models, adjusted for GFR, age, gender and inflammatory markers including C-reactive protein (CRP), albumin, ferritin, IL-6, IL-8 and TNF-α, anemia predicted mental components of SF-36 (SF-12 mental component (p = 0.02) and role emotional (p = 0.04)) but not physical components (SF-12 physical component (p > 0.05) and physical function (p > 0.05), supporting the multifactorial nature of reduced HRQOL in anemic patients. CONCLUSIONS: Reduced HRQOL in anemic patients is likely related to both anemic and inflammatory status. Prospective studies will be needed to evaluate whether modulating the inflammatory state independent of changes in the hemoglobin concentration improves physical components of HRQOL.

Endothelial progenitors encapsulated in bioartificial niches are insulated from systemic cytotoxicity and are angiogenesis competent.

Intrinsic stem cells (SC) participate in tissue remodeling and regeneration in various diseases and following toxic insults. Failure of tissue regeneration is in part attributed to lack of SC protection from toxic stress of noxious stimuli, thus prompting intense research efforts to develop strategies for SC protection and functional preservation for in vivo delivery. One strategy is creation of artificial SC niches in an attempt to mimic the requirements of endogenous SC niches by generating scaffolds with properties of extracellular matrix. Here, we investigated the use of hyaluronic acid (HA) hydrogels as an artificial SC niche and examined regenerative capabilities of encapsulated embryonic endothelial progenitor cells (eEPC) in three different in vivo models. Hydrogel-encapsulated eEPC demonstrated improved resistance to toxic insult (adriamycin) in vitro, thus prompting in vivo studies. Implantation of HA hydrogels containing eEPC to mice with adriamycin nephropathy or renal ischemia resulted in eEPC mobilization to injured kidneys (and to a lesser extent to the spleen) and improvement of renal function, which was equal or superior to adoptively transferred EPC by intravenous infusion. In mice with hindlimb ischemia, EPC encapsulated in HA hydrogels dramatically accelerated the recovery of collateral circulation with the efficacy superior to intravenous infusion of EPC. In conclusion, HA hydrogels protect eEPC against adriamycin cytotoxicity and implantation of eEPC encapsulated in HA hydrogels supports renal regeneration in ischemic and cytotoxic (adriamycin) nephropathy and neovascularization of ischemic hindlimb, thus establishing their functional competence and superior capabilities to deliver stem cells stored in and released from this bioartificial niche.

Postobstructive regeneration of kidney is derailed when surge in renal stem cells during course of unilateral ureteral obstruction is halted.

Unilateral ureteral obstruction (UUO), a model of tubulointerstitial scarring (TIS), has a propensity toward regeneration of renal parenchyma after release of obstruction (RUUO). No information exists on the contribution of stem cells to this process. We performed UUO in FVB/N mice, reversed it after 10 days, and examined kidneys 3 wk after RUUO. UUO resulted in attenuation of renal parenchyma. FACS analysis of endothelial progenitor (EPC), mesenchymal stem (MSC) and hematopoietic stem (HSC) cells obtained from UUO kidneys by collagenase-dispersed single-cell suspension showed significant increase in EPC, MSC, and HSC compared with control. After RUUO cortical parenchyma was nearly restored, and TIS score improved by 3 wk. This reversal process was associated with return of stem cells toward baseline level. When animals were chronically treated with nitric oxide synthase (NOS) inhibitor at a dose that did not induce hypertension but resulted in endothelial dysfunction, TIS scores were not different from control UUO, but EPC number in the kidney decreased significantly; however, parenchymal regeneration in these mice was similar to control. Blockade of CXCR4-mediated engraftment resulted in dramatic worsening of UUO and RUUO. Similar results were obtained in caveolin-1-deficient but not -overexpressing mice, reflecting the fact that activation of CXCR4 occurs in caveolae. The present data show increase in EPC, HSC, and MSC population during UUO and a tendency for these cells to decrease to control level during RUUO. These processes are minimally affected by chronic NOS inhibition. Blockade of CXCR4-stromal cell-derived factor-1 (SDF-1) interaction by AMD3100 or caveolin-1 deficiency significantly reduced the UUO-associated surge in stem cells and prevented parenchymal regeneration after RUUO. We conclude that the surge in stem cell accumulation during UUO is a prerequisite for regeneration of renal parenchyma.

Epac-1 activator 8-O-cAMP augments renoprotective effects of syngeneic [corrected] murine EPCs in acute ischemic kidney injury.

Endothelial progenitor cells (EPCs) protect kidneys from acute ischemic damage. The aim of this study was to identify "treatment parameters" that optimize an EPC-based therapy of acute ischemic renal failure. Male C57BL/6N mice underwent unilateral nephrectomy with simultaneous contralateral renal artery clamping for 30, 35, and 40 min. Tagged murine EPCs were systemically injected at the time of reperfusion. In some experiments, EPCs were pretreated with the Epac (exchange protein directly activated by cAMP-1) activator 8-pCPT-2'-O-Me-cAMP (Epac-1 Ac) and the integrin binding antagonist cyclic Arg-Gly-Asp peptide (cRGD). Injections of 10(6) EPCs after 30 and 35 min of renal ischemia protected animals from acute renal failure. The same effect occurred with 0.5 x 10(6) EPCs after a 35-min period of ischemia. If ischemia lasted for 40 min, 0.5 x 10(6) cells mice did not prevent acute renal failure. To analyze whether EPC integrin receptor activation would modify the cells' renoprotective activity, EPCs were pretreated with Epac-1 Ac. Such animals did not develop acute renal failure, even if ischemia lasted for 40 min. This effect was negated if the cells were pretreated with both Epac-1 Ac and cRGD. In kidneys from those animals medullopapillary EPCs were significantly accumulated. In vitro Epac-1 Ac preactivation of EPCs did not increase the overall expression intensity but induced a redistribution of beta(1)-integrins toward the cell membranes. We conclude that EPC pretreatment with the integrin receptor activator 8-pCPT-2'-O-Me-cAMP augments the anti-ischemic potential of the cells.

Urine proteomics--prospects for future diagnostics.

This brief overview of the potential diagnostic, prognostic and pathophysiological value of studies into the urine proteome describes hypothesis-driven investigations of individual proteins and proteome-wide search for urinary biomarkers of various diseases and their progression. It is intended to illustrate the recent progress in the area of urine proteomics and proselytize for the promise of this centuries-old technique of uroscopy, yet to reveal its secrets, using modem approaches.

Characterization of urinary peptide biomarkers of acute rejection in renal allografts.

We previously demonstrated that 4.7 kDa and 4.4 kDa peptides are useful in diagnosing acute rejection in renal transplant recipients. The aim of this study was to characterize these polypeptides and assess their potential as biomarkers. The polypeptides were identified as human beta-Defensin-1 (4.7 kDa) and alpha-1-antichymotrypsin (4.4 kDa), by tandem mass spectrometry and ProteinChip immunoassay. The urinary abundance of both polypeptides, assessed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), revealed a reduction in beta-Defensin-1 while alpha-1-antichymotrypsin increased in patients with rejection (p < 0.05) compared with clinically stable transplants. The area under the curve (AUC) for the receiver operator characteristic (ROC) curve for the diagnosis of rejection for the ratio of both peptides combined was 0.912. Longitudinal analysis confirmed a reduction in beta-Defensin-1 with a reciprocal increase in alpha-1-antichymotrypsin as rejection developed. The difference in urinary beta-Defensin-1 levels quantified by radioimmunoassay was 176.8 +/- 122.3 pg/mL in stable patients compared with 83.2 +/- 52.2 pg/mL in patients with acute rejection, with an ROC AUC of 0.749 (p < 0.01). Immunohistochemistry (IHC) confirmed reduced beta-Defensin-1 expression in the renal parenchyma of patients experiencing acute rejection. In conclusion, the ratio of beta-Defensin-1 and alpha-1-antichymotrypsin excretion in the urine is a novel, potentially useful candidate biomarkers of acute rejection.

Normal distribution and medullary-to-cortical shift of Nestin-expressing cells in acute renal ischemia.

Nestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 microm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration.

Meeting report: ISN forefronts in nephrology on endothelial biology and renal disease: from bench to prevention.

This ISN-sponsored Forefront in Nephrology meeting, which has brought together 120 scientists from 21 countries, has been concerned with various aspects of endothelial function and dysfunction and their contribution to progression of chronic kidney disease and/or its cardiovascular complications. The following themes were discussed in great depth: (1) phenotypical changes in the vascular endothelium - permeability, senescence, and apoptosis; (2) regulation of endothelial nitric oxide (NO) synthase function - caveolar and shear stress mechanisms, epigenetic regulation, S-nitrosylation, and Rho-kinase regulation; (3) oxidative stress and hypoxia-induced changes; (4) organellar dysfunction - lysosomes, mitochondria, and endoplasmic reticulum; (5) NO-independent mechanisms of vasomotion - epoxides, heme oxygenase-1 and carbon monoxide, thromboxane, tumor necrosis factor-alpha, and uric acid; (6) endothelial crosstalk with podocytes, monocytes, smooth muscle cells, and platelets; (7) candidate clinical biomarkers of endothelial dysfunction - functional testing of macro- and micro-vascular functions, surrogate markers, circulating detached endothelial cells, and endothelial precursor cells; and culminated in Round Table discussion on the diagnosis of endothelial dysfunction and its treatment options. In conclusion, this meeting has focused on several key problems of endothelial cell pathobiology relevant to chronic kidney disease.

Laser Doppler flowmetry detection of endothelial dysfunction in end-stage renal disease patients: correlation with cardiovascular risk.

Prediction of cardiovascular (CV) complications represents the Achilles' heel of end-stage renal disease. Surrogate markers of endothelial dysfunction have been advocated as predictors of CV risk in this cohort of patients. We have recently adapted a noninvasive laser Doppler flowmetry (LDF) functional testing of endothelium-dependent microvascular reactivity and demonstrated that end-stage renal disease patients are characterized by profound alterations in thermal hyperemic responsiveness. We hypothesized that such functional assessment of the cutaneous microcirculation may offer a valid, noninvasive test of the severity of endothelial dysfunction and CV risk. To test this hypothesis, we performed a cross-sectional study, in which we compared LDF measurements to conventional risk factors, and performed a pilot longitudinal study. LDF studies were performed in 70 patients and 33 controls. Framingham and Cardiorisk scores were near equivalent for low-risk patients, but more divergent as risk increased. C reactive protein (CRP) levels and LDF parameters (amplitude of thermal hyperemia (TH), area under the curve of TH) showed significant abnormality in high-risk vs low-risk patients calculated using either Framingham or Cardiorisk scores. Patients who had abnormal LDF parameters showed increased CV mortality, however, had similar risk assessments (Framingham, Cardiorisk, CRP, and homocysteine) to those with unimpaired LDF tracings. In conclusion, LDF parameters of microvascular reactivity offer a sensitive characterization of endothelial dysfunction, which may improve CV risk assessment through incorporation into the Framingham or Cardiorisk algorithm.

Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation.

Intimal infiltration by monocytes and accumulation of lipids represent a critical step in the formation of fatty streaks during atherogenesis. Because elevated plasma levels of asymmetric dimethylarginine (ADMA), a potent nitric oxide (NO) synthase (NOS) inhibitor, are prevalent in diverse cardiovascular diseases, the goal of this study was to examine the contribution of NO deficiency to macrophage lipid accumulation. Inhibition of NO synthesis in PMA-primed human monocytic leukemia HL-60 cells resulted in a twofold increase in expression of the receptor for oxidized LDL (OxLDL), termed the lectin-like OxLDL receptor (LOX-1). Blockade of inducible NOS in activated macrophages resulted in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-OxLDL accumulation and imparted macrophages with a foamy appearance as detected with oil-red O lipid staining. ADMA (15 microM) or N(G)-nitro-l-arginine methyl ester (l-NAME, 300 microM), both of which suppress inducible NOS activity, increased oil-red staining 1.9- and 2.8-fold, respectively. Macrophages treated with ADMA or l-NAME showed a 2.4-fold increase in accumulation of DiI-OxLDL. To examine the role of LOX-1 in this process, we used small interfering RNA (siRNA) duplex-mediated LOX-1 gene silencing. LOX-1 expression was suppressed twofold by siRNA as shown by Western blot analysis. This suppression was associated with a two- to fourfold decrease in DiI-OxLDL uptake as identified by fluorescence microscopy and decreased oil-red O staining by activated macrophages. In conclusion, accumulation of ADMA (a competitive inhibitor of NOS) in patients with chronic renal failure may be responsible for upregulation of LOX-1 receptor and increased OxLDL uptake, thus contributing to lipidosis and foam cell formation. The data illustrate an additional nonendothelial mode of antiatherogenic action of NO: prevention of LOX-1 induction and lipid accumulation by macrophages.

Upregulation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells by nitric oxide deficiency.

Endothelial cell dysfunction (ECD) is emerging as a common denominator for diverse cardiovascular abnormalities associated with inhibition of endothelial nitric oxide (NO) synthase (eNOS). Elevated levels of asymmetric dimethylarginine (ADMA), a potent eNOS inhibitor, are common in renal failure and may contribute to ECD. Through DNA microarray screening of genes modulated in human umbilical vein endothelial cells (HUVEC) by N(G)-nitro-l-arginine methyl ester (l-NAME), we found a 1.8-fold increase in low-density lipoprotein receptor-1 (LOX-1) expression. LOX-1 is a major endothelial receptor for oxidized low-density lipoproteins (OxLDL) and is assumed to play a role in the initiation and progression of atherosclerosis. Here, we confirmed the upregulation of LOX-1 mRNA and protein level by quantitative RT-PCR and Western blot analysis. Increased expression of LOX-1 was associated with the accumulation of DiI-labeled OxLDL (DiI-OxLDL) in ADMA- and l-NAME-pretreated HUVEC. To evaluate the contribution of LOX-1 in ADMA-induced accumulation of OxLDL by HUVEC, we used the competitive receptor inhibitor, soluble LOX-1. Treatment of HUVEC with soluble LOX-1 was associated with an approximately two- to threefold inhibition of DiI-OxLDL uptake in l-NAME- or ADMA-treated HUVEC. In conclusion, ADMA- or l-NAME-induced NO deficiency leads to the increased expression of LOX-1 mRNA and protein in HUVEC, which in turn results in the accumulation of OxLDL. Competition with LOX-1-soluble extracellular domain reduces OxLDL accumulation. In summary, elevated ADMA levels, i.e., in patients with renal failure, may be responsible for endothelial accumulation of OxLDL via upregulated LOX-1 receptor, thus contributing to endothelial lipidosis and dysfunction.

Oxidative and nitrosative stress in acute renal ischemia.

Generation of reactive oxygen species and nitric oxide in hypoxia-reperfusion injury may form a cytotoxic metabolite, peroxynitrite, which is capable of causing lipid peroxidation and DNA damage. This study was designed to examine the contribution of oxidative and nitrosative stress to the renal damage in ischemic acute renal failure (iARF). iARF was initiated in rats by 45-min renal artery clamping. This resulted in lipid peroxidation, DNA damage, and nitrotyrosine modification confirmed both by Western and immunohistochemical analyses. Three groups of animals were randomly treated with an inhibitor of inducible nitric oxide synthase (NOS), L-N(6)-(1-iminoethyl)lysine (L-Nil), cell-permeable lecithinized superoxide dismutase (SOD), or both. Each treatment resulted in amelioration of renal dysfunction, as well as reduced nitrotyrosine formation, lipid peroxidation, and DNA damage, thus suggesting that peroxynitrite rather than superoxide anion is responsible for lipid peroxidation and DNA damage. Therefore, in a separate series of experiments, a scavenger of peroxynitrite, ebselen, was administered before the reperfusion period. This treatment resulted in a comparable degree of amelioration of iARF. In conclusion, the present study provides the first attempt to elucidate the role of peroxynitrite in initiation of the cascade of lipid peroxidation and DNA damage to ischemic kidneys. The results demonstrate that L-Nil, lecithinized SOD, and ebselen treatments improve renal function due to their suppression of peroxynitrite production or its scavenging, consequently preventing lipid peroxidation and oxidative DNA damage.

Plasmin-dependent and -independent effects of plasminogen activators and inhibitor-1 on ex vivo angiogenesis.

Plasminogen activator (PA) inhibitor-1 (PAI-1) has been recognized as a surrogate marker of endothelial dysfunction in diseases associated with impaired angiogenesis, including atherosclerosis, diabetic vasculopathy, and nephropathy. To establish the necessary and sufficient components of the PA system [PAI-1, urokinase-type PA (uPA), or tissue-type PA (tPA), and plasminogen (Plg)] for angiogenesis, we examined angiogenic competence of vascular explant cultures obtained from mice deficient in PAI-1, tPA, uPA, and Plg. To gain insight into the requirement for different matrix-degrading systems during endothelial cell migration across plasmin-degradable basement membranes compared with profibrotic areas containing plasmin-nondegradable collagen, we contrasted vascular sprouting in collagen with Matrigel lattices. PAI-1(-/-) vessels showed an increased capillary sprouting in both collagen and Matrigel. Deficiency of uPA significantly reduced the rate of sprouting, whereas tPA(-/-) vessels showed a profound inhibition of capillary sprouting. The Plg(-/-) vessels failed to sprout, a defect that was restored not only by exogenous Plg, but also by the addition of PAs; a nonproteolytic effect of tPA was observed in Matrigel. Zymography revealed no differences in the activity of metalloproteinase (MMP)-2 and -9 in wild-type and PAI-1(-/-) vessels, but demonstrated reduced MMP-9 activity in all angiogenesis-deficient vessels. In summary, 1) PAI-1 by itself is a modest inhibitor of endothelial sprouting, 2) tPA and Plg are indispensable for angiogenesis in this model, 3) Plg is not the only substrate for PAs, and 4) the activity of MMP-9 is undetectable in explant cultures from tPA and Plg knockout mice.

Delayed branching of endothelial capillary-like cords in glycated collagen I is mediated by early induction of PAI-1.

Development of micro- and macrovascular disease in diabetes mellitus (DM) warrants a thorough investigation into the repertoire of endothelial cell (EC) responses to diabetic environmental cues. Using human umbilical vein EC (HUVEC) cultured in three-dimensional (3-D) native collagen I (NC) or glycated collagen I (GC), we observed capillary cord formation that showed a significant reduction in branching when cells were cultured in GC. To gain insight into the molecular determinants of this phenomenon, HUVEC subjected to GC vs. NC were studied using a PCR-selected subtraction approach. Nine different genes were identified as up- or downregulated in response to GC; among those, plasminogen activator inhibitor-1 (PAI-1) mRNA was found to be upregulated by GC. Western blot analysis of HUVEC cultured on GC showed an increase in PAI-1 expression. The addition of a neutralizing anti-PAI-1 antibody to HUVEC cultured in GC restored the branching pattern of formed capillary cords. In contrast, supplementation of culture medium with the constitutively active PAI-1 reproduced defective branching patterns in HUVEC cultured in NC. Ex vivo capillary sprouting in GC was unaffected in PAI-1 knockout mice but was inhibited in wild-type mice. This difference persisted in diabetic mice. In conclusion, the PCR-selected subtraction technique identified PAI-1 as one of the genes characterizing an early response of HUVEC to the diabetic-like interstitial environment modeled by GC and responsible for the defective branching of endothelial cells. We propose that an upregulation of PAI-1 is causatively linked to the defective formation of capillary networks during wound healing and eventual vascular dropout characteristic of diabetic nephropathy.

The concept of cellular "fight-or-flight" reaction to stress.

As animals respond to environmental stress with a set of default reactions described as the "fight-or-flight" response, so do epithelial and endothelial cells when they are confronting stressors in their microenvironment. This review will summarize a growing body of data suggesting the existence of a set of stereotypical cellular reactions to stress, provide some examples of diseases that are characterized by excessive flight reactions, describe the cellular mechanisms whereby the fight-or-flight reaction is accomplished, as well as cellular mechanisms triggering either fight or flight. It is proposed that cell-matrix adhesion is a sensitive indicator of the severity of stress. This indicator is interfaced with several default programs for cellular survival or death, thus dictating the fate of the cell. Some diagnostic and therapeutic applications of the concept, presently used and potentially useful, are outlined. The essential feature of this concept is its ability to categorize cellular events in terms of either type of default reaction, predict the details of each, and potentially exploit them clinically.

Workshop: endothelial cell dysfunction leading to diabetic nephropathy : focus on nitric oxide.

Clinical manifestations of diabetic nephropathy are an expression of diabetic microangiopathy. This review revisits the previously proposed Steno hypothesis and advances our hypothesis that development of endothelial cell dysfunction represents a common pathophysiological pathway of diabetic complications. Specifically, the ability of glucose to scavenge nitric oxide is proposed as the initiation phase of endothelial dysfunction. Gradual accumulation of advanced glycated end products and induction of plasminogen activator inhibitor-1, resulting in the decreased expression of endothelial nitric oxide synthase and reduced generation of nitric oxide, are proposed to be pathophysiologically critical for the maintenance phase of endothelial dysfunction. The proposed conceptual shift toward the role of endothelial dysfunction in diabetic complications may provide new strategies for their prevention.