Biomedical subjects

Hui Y Lan

Publications and source records attributed to Hui Y Lan.

31 records · Page 2Linked to original sources

PubMed2004

Advanced glycation end products induce tubular epithelial-myofibroblast transition through the RAGE-ERK1/2 MAP kinase signaling pathway.

Advanced glycation end products (AGEs) have been shown to play a role in tubular epithelial-myofibroblast transdifferentiation (TEMT) in diabetic nephropathy, but the intracellular signaling pathway remains unknown. We report here that AGEs signal through the receptor for AGEs (RAGE) to induce TEMT, as determined by de novo expression of a mesenchymal marker (alpha-smooth muscle actin, alpha-SMA) and loss of epithelial marker (E-cadherin), directly through the MEK1-ERK1/2 MAP kinase pathway, which is TGF-beta independent. This is supported by the following findings: AGEs induced de novo alpha-SMA mRNA expression as early as 2 hours followed by a loss of E-cadherin before TGF-beta mRNA expression at 24 hours and occurred in the absence of TGF-beta and AGE-induced activation of ERK1/2 MAP kinase at 15 minutes and TEMT at 24 hours were completely blocked by a neutralizing RAGE antibody, a soluble RAGE receptor, an ERK1/2 MAP kinase inhibitor (PD98059), and DN-MEK1, but not by a neutralizing TGF-beta antibody. Thus, this study demonstrates that AGEs activate the RAGE-ERK1/2 MAP kinase pathway to mediate the early TEMT process. The findings from this study suggest that targeting the RAGE or the ERK MAP kinase pathway may provide new therapeutic strategies for diabetic nephropathy and shed new light on the pathogenesis of diabetic nephropathy.

Actins↗

PubMed2004

TGF-beta induces proangiogenic and antiangiogenic factors via parallel but distinct Smad pathways.

BACKGROUND: Angiogenesis has a key role in numerous disease processes. One of the most important angiogenic factors is vascular endothelial growth factor (VEGF-A), whereas thrombospondin-1 (TSP-1) is a major antiangiogenic factor. Recent studies have shown that VEGF-A as well as TSP-1 is regulated by transforming growth factor-beta1 (TGF-beta1), but the mechanism remains unclear. METHODS: We examined the role of TGF-beta1 and its signaling pathways in mediating expression of these two molecules. Rat proximal tubular cells (NRK52E) were stimulated with TGF-beta1 to induce VEGF-A and TSP-1 synthesis. To clarify roles of receptor-activated Smads (R-Smads), we blocked Smad signaling using overexpression of the inhibitory Smad, Smad7, and by using fibroblasts from wild-type or knockout mice. To confirm the antiantigenic role of Smads, soluble Flt-1 regulation in response to TGF-beta1 was also examined. In addition, the effect of conditioned media from NRK52E and Smad knockout cells was examined on endothelial cell proliferation. RESULTS: Induction of VEGF-A and TSP-1 by TGF-beta1 in NRK52E cells was associated with activation of pathway-restricted R-Smads (Smad2 and 3) and blocking these Smads by overexpression of Smad7 blocked their induction. By using of Smad knockout cells, Smad3 was shown to have a key role in the stimulation of VEGF-A expression whereas Smad2 was critical for TSP-1 expression. Consistent with the hypothesis that Smad2 has an antiangiogenic function, we also demonstrated that Smad2, but not Smad3, mediated the expression of VEGF-A antagonist, soluble VEGF-A receptor sFlt-1, in response to TGF-beta1. Conditioned media from NRK52E, which was stimulated by TGF-beta1 for 24 hours, did not induce endothelial cell proliferation. However, conditioned media from Smad2 knockout induced endothelial cell proliferation, whereas endothelial cell proliferation was inhibited by Smad3 knockout-derived conditioned media. CONCLUSION: R-Smads have distinct roles in mediating the expression of pro- and antiangiogenic growth factors in response to TGF-beta1.

Animals↗

PubMed2003

Uric acid stimulates monocyte chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-activated protein kinase and cyclooxygenase-2.

Previous studies have reported that uric acid stimulates vascular smooth muscle cell (VSMC) proliferation in vitro. We hypothesized that uric acid may also have direct proinflammatory effects on VSMCs. Crystal- and endotoxin-free uric acid was found to increase VSMC monocyte chemoattractant protein-1 (MCP-1) expression in a time- and dose-dependent manner, peaking at 24 hours. Increased mRNA and protein expression occurred as early as 3 hours after uric acid incubation and was partially dependent on posttranscriptional modification of MCP-1 mRNA. In addition, uric acid activated the transcription factors nuclear factor-kappaB and activator protein-1, as well as the MAPK signaling molecules ERK p44/42 and p38, and increased cyclooxygenase-2 (COX-2) mRNA expression. Inhibition of p38 (with SB 203580), ERK 44/42 (with UO126 or PD 98059), or COX-2 (with NS398) each significantly suppressed uric acid-induced MCP-1 expression at 24 hours, implicating these pathways in the response to uric acid. The ability of both n-acetyl-cysteine and diphenyleneionium (antioxidants) to inhibit uric acid-induced MCP-1 production suggested involvement of intracellular redox pathways. Uric acid regulates critical proinflammatory pathways in VSMCs, suggesting it may have a role in the vascular changes associated with hypertension and vascular disease.

Animals↗

PubMed2003

Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease.

While it is thought that advanced glycation end products (AGEs) act by stimulating transforming growth factor (TGF)-beta to mediate diabetic injury, we report that AGEs can activate TGF-beta signaling, Smads, and mediate diabetic scarring directly and independently of TGF-beta. AGEs activate Smad2/3 in renal and vascular cells at 5 min, peaking over 15-30 min before TGF-beta synthesis at 24 h and occurs in TGF-beta receptor I and II mutant cells. This is mediated by RAGE and ERK/p38 mitogen-activated protein kinases (MAPKs). In addition, AGEs also activate Smads at 24 h via the classic TGF-beta-dependent pathway. A substantial inhibition of AGE-induced Smad activation and collagen synthesis by ERK/p38 MAPK inhibitors, but not by TGF-beta blockade, suggests that the MAPK-Smad signaling crosstalk pathway is a key mechanism in diabetic scarring. Prevention of AGE-induced Smad activation and collagen synthesis by overexpression of Smad7 indicates that Smad signaling may play a critical role in diabetic complications. This is further supported by the findings that activation of Smad2/3 in human diabetic nephropathy and vasculopathy is associated with local deposition of AGEs and up-regulation of RAGE. Thus, AGEs act by activating Smad signaling to mediate diabetic complications via both TGF-beta-dependent and -independent pathways, shedding new light on the pathogenesis of diabetic organ injury.

Cells, Cultured↗

PubMed2003

Role of TGF-beta signaling in extracellular matrix production under high glucose conditions.

BACKGROUND: Hyperglycemia has been shown to play an important role in diabetic renal and vascular complications. Some studies show that high glucose may mediate diabetic complications by stimulating extracellular matrix (ECM) production. We hypothesize that this may be mediated by activating transforming growth factor-beta (TGF-beta)/Smads signaling. METHODS: Renal and vascular cells were cultured under high glucose conditions in the presence or absence of a neutralizing TGF-beta antibody and examined for activation of Smad signaling and collagen production. The regulating role of Smad signaling in high glucose-induced collagen synthesis was determined by inducing overexpression of the inhibitory Smad7 in a stable Smad7-expressing tubular cell line. RESULTS: Activation of Smad signaling, as evidenced by Smad2 and Smad3 nuclear translocation and phosphorylation, was found in renal and vascular cells at 24 hours after high glucose stimulation (up to 55% increased). This was associated with de novo synthesis of collagen I at day 3 by all cell types. High glucose-induced activation of Smad signaling and collagen synthesis were TGF-beta-dependent since these were associated with a significant increase in TGF-beta production at 24 hours (P < 0.01) and were blocked by a neutralizing TGF-beta antibody. Importantly, overexpression of Smad7 resulted in marked inhibition of high glucose-induced Smad2 and Smad3 activation and type I collagen synthesis, suggesting that Smad signaling is a key pathway in high glucose-mediated renal and vascular scarring. CONCLUSION: High glucose acts by activating the TGF-beta dependent Smad signaling pathway to stimulate collagen synthesis by renal and vascular cells. Smad signaling plays a critical role in regulating high-glucose-mediated diabetic renal and vascular complications.

Animals↗

PubMed2003

Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule cells.

PURPOSE OF REVIEW: Emerging evidence suggests that tubular epithelial-myofibroblast transdifferentiation is an important event in renal tubulointerstitial fibrosis. This review describes the recent findings in the context of the tubular epithelial-myofibroblast transdifferentiation process and discusses the possible mechanisms involved. RECENT FINDINGS: Tubular epithelial-myofibroblast transdifferentiation is a complex process involving disruption of polarized tubular epithelial cell morphology into cells with spindle-shaped mesenchymal morphology, formation of actin stress fibers, loss of cell-cell adhesions through downregulation of E-cadherin, destruction of basement membrane, and increased cell migration and invasion. This phenotypic transition has also been recently reported in human glomerulonephritis with progressive tubulointerstitial fibrosis. Transforming growth factor-beta is a key fibrogenic growth factor that regulates tubular epithelial-myofibroblast transdifferentiation, which is counter-regulated by hepatocyte growth factor. In addition, basic fibroblast growth factor, advanced glycation end products, and angiotensin II have also been reported to induce the process. Importantly, the recent discovery of transforming growth factor-beta/Smad signaling has allowed the delineation of the intracellular mechanisms of tubular epithelial-myofibroblast transdifferentiation. Indeed, Smad signaling is a key pathway whereby transforming growth factor-beta and angiotensin II induce tubular epithelial-myofibroblast transdifferentiation in vitro. This involves the activation of transforming growth factor-beta receptor-associated Smad2 and is inhibited by an inhibitory Smad protein, Smad7. Thus, Smad signaling plays a critical role in tubular epithelial-myofibroblast transdifferentiation. SUMMARY: Renal myofibroblasts may be derived from tubular epithelial cells by a process of tubular epithelial-myofibroblast transdifferentiation. Transforming growth factor-beta signals through Smads to positively or negatively regulate this process. Blockade of this process by either hepatocyte growth factor or targeting the Smad signaling pathway may provide novel therapeutic strategies to combat renal fibrosis.

Actins↗

PubMed2003

Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.

TGF-beta is a key mediator in renal fibrosis. Kidney-targeted gene therapy with anti-TGF-beta strategies is expected to have therapeutic potential, but this has been hampered by concerns over the safety and practicability of viral vectors and the inefficiency of nonviral transfection techniques. The present study explored the potential role of TGF-beta/Smad signaling in renal fibrosis in vivo and developed a safe and effective gene therapy to specifically block TGF-beta signaling and renal fibrosis in a rat unilateral ureteral obstruction (UUO) model by transferring a doxycycline-regulated Smad7 gene or control empty vectors using an ultrasound-microbubble (Optison)-mediated system. The Smad7 transgene expression was tightly controlled by addition of doxycycline in the daily drinking water. Groups of six rats were sacrificed at day 7, and the transfection rate, Smad7 transgene expression, and tubulointerstitial fibrosis including alpha-smooth muscle actin and collagen matrix mRNA and protein expression were determined. Compared with the non-ultrasound treatment, the combination of ultrasound with Optison largely increased the transfection rate of FITC-ODN and Smad7 transgene expression up to a 1000-fold, and this was found in all kidney tissues. Compared with normal rats, Smad7 expression within the UUO kidney was significantly reduced, and this was associated with up to a sixfold increase in Smad2 and Smad3 activation and severe tubulointerstitial fibrosis. In contrast, treatment with inducible Smad7 resulted in a fivefold increase in Smad7 expression with complete inhibition of Smad2 and Smad3 activation and tubulointerstitial fibrosis in terms of tubulointerstitial myofibroblast accumulation (85% downward arrow ) and collagen I and III mRNA and protein expression (60 to 70% downward arrow ). In conclusion, the ultrasound-mediated inducible Smad7 gene transfer is a safe, effective, and controllable gene therapy. TGF-beta-mediated renal fibrosis is regulated positively by Smad2/3, but negatively by Smad7. Target blockade of TGF-beta/Smad signaling by expression of Smad7 may provide a new therapeutic potential for renal fibrosis.

Air↗

PubMed2003

Chymase is upregulated in diabetic nephropathy: implications for an alternative pathway of angiotensin II-mediated diabetic renal and vascular disease.

Angiotensin II (AngII) has been shown to play a critical role in diabetic nephropathy and vasculopathy. Although it is well recognized that an angiotensin-converting enzyme (ACE)-dependent AngII-generating system is a major source of intrarenal AngII production, it is here reported that the chymase-dependent AngII-generating system is upregulated in the human diabetic kidney. This becomes particularly strong in those with hypertension. In the normal kidney, while ACE was constitutively expressed by most kidney cells, chymase was weakly expressed by mesangial cells (MC) and vascular smooth muscle cells (VSMC) only. In the diabetic kidney, while ACE expression was significantly upregulated (1 to 3-fold) by tubular epithelial cells (TEC) and infiltrating mononuclear cells, there was also markedly increased chymase expression (10 to 15-fold) by both MC and VSMC, with strong deposition in the collagen-rich extracellular matrix including both diffuse and nodular glomerulosclerosis, tubulointerstitial fibrosis, and vascular sclerosis. Interestingly, while ACE expression showed no difference in patients with or without hypertension, upregulation of chymase in hypertensive patients was much stronger than that seen in those without hypertension (4 to 7-fold, P < 0.001). Correlation analysis showed that, in contrast to the ACE expression, upregulation of chymase correlated significantly with the increase in BP and the severity of collagen matrix deposition within the glomerulus, tubulointerstitium, and arterial walls (all with P < 0.001). In conclusion, the present study demonstrates that chymase, as an alternative AngII-generating enzyme, is markedly upregulated in the diabetic kidney and may be associated with the development of diabetic/hypertensive nephropathy. In addition, differential expression of ACE and chymase in the diabetic kidney indicates that both ACE and chymase may be of equal importance for AngII-mediated diabetic nephropathy and vascular disease. Results from this study suggest that blockade of both AngII-generating pathways may provide additional beneficial effect on diabetic nephropathy.

Adult↗

PubMed2003

Expression of macrophage migration inhibitory factor in acute ischemic myocardial injury.

Macrophage migration inhibitory factor (MIF) is a key mediator in inflammatory or immune-mediated diseases, although its role in heart diseases is unknown. This study investigated the expression of MIF in the myocardium in the development of acute myocardial infarction (AMI). By use of immunohistochemistry, Western blotting, RT-PCR, and in situ hybridization, the gene and protein expression of MIF in the heart at 6 hr, 1 day, 3 days, 1 week, and 2 weeks after AMI was studied. In both normal and sham-operated rats, MIF mRNA and protein were expressed constitutively at low levels by the myocytes. By contrast, MIF mRNA was rapidly upregulated by the surviving myocytes in the infarcted region and, to a lesser extent, the non-infarcted region, accounting for a sevenfold increase at 6 hr after AMI (p<0.001). This was followed by a fourfold increase in MIF protein expression at day 1 after AMI (p<0.05). Macrophages were found accumulated in the infarcted region, being significant at day 1 (p<0.01) and progressive increased over the 2-week time course (p<0.01) in which MIF was found expressed in these cells. The results indicated that the infiltrating macrophages and myocytes were sources of MIF in the infarcted region. The latter cells became activated and involved in the amplification of inflammatory response in AMI. Therefore, upregulation of myocardial MIF may contribute to macrophage accumulation in the infarcted region and their pro-inflammatory role may participate in the myocyte damage seen in AMI.

Animals↗

PubMed2002

Effects of cyclosporine in osteopontin null mice.

BACKGROUND: Osteopontin (OPN) is a macrophage adhesive and cell survival factor that is up-regulated in tubules in tubulointerstitial disease. We have previously reported that rats with cyclosporine (CsA) nephropathy have increased tubular osteopontin that correlates with the infiltration of macrophages and interstitial fibrosis. This study tested the hypothesis that the absence of OPN would ameliorate CsA nephropathy. METHODS: OPN knockout (-/-) and wild type (+/+) mice were fed a low salt diet (Na+ 0.01%) for one week and then received daily CsA injections (30 mg/kg, SC) until sacrifice at two weeks. Afferent arteriolopathy, tubulointerstitial injury, macrophage infiltration, collagen III deposition, transforming growth factor-beta (TGF-beta) expression, and tubular and interstitial cell proliferation and apoptosis were evaluated. RESULTS: Wild type mice developed early features of CsA nephropathy, with arteriolar hyalinosis and cortical and tubulointerstitial fibrosis. Despite comparable CsA levels, OPN-/- mice had less arteriolopathy (15 vs. 24%, P < 0.05), a 20% reduction in cortical macrophage infiltration (P < 0.05), and 20% reduction in interstitial collagen deposition (P < 0.05). OPN-/- mice also showed less cortical interstitial cell proliferation but no differences in tubular cell proliferation or apoptosis. OPN+/+ mice also developed some neurotoxicity, consisting of ataxia, and this was associated with increased mortality at two weeks. CONCLUSION: OPN partially mediates arteriolopathy, early macrophage recruitment and fibrosis in murine CsA nephropathy. OPN also may be involved in CsA associated neurotoxicity.

Animals↗

PubMed2002

Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation.

It has been shown that transforming growth factor-beta (TGF-beta) is a potent mediator in renal fibrosis and that Smad proteins are critical intracellular mediators in TGF-beta signaling. It is here reported that TGF-beta mediates renal fibrogenesis in tubular epithelial cells (TEC) in association with the activation of Smad2 and that overexpression of Smad7 blocks this fibrotic process. Using a normal rat kidney tubular epithelial cell line (NRK52E), it was determined that TGF-beta1 induces Smad2 phosphorylation and nuclear localization in both a dose- and time-dependent manner. The activation of Smad2 was evident at 5 min (20%), peaked at 15 to 30 min (85%), and declined to baseline levels by 2 h (5 to 10%). This was associated with de novo expression of collagens I, III, and IV and the transformation of TEC into a "myofibroblast" phenotype with de novo expression of alpha-smooth muscle actin (alpha-SMA) and with the loss of E-cadherin (>50%). To investigate a negative regulatory role of Smad7 in renal fibrosis, the Smad 7 gene was stably transfected and its expression was tightly controlled by doxycycline into NRK52E cells. Overexpression of Smad7 induced by doxycycline results in marked inhibition of TGF-beta-induced Smad2 activation (90% downward arrow) with the prevention of collagen synthesis and myofibroblast transformation. Thus, Smad2 activation occurs in the fibrogenic response of TEC to TGF-beta, and this process is blocked by overexpression of Smad7. This indicates that Smad signaling is a key pathway of TGF-beta-mediated renal fibrosis and suggests that treatments targeting the inactivation of Smad2 by overexpression of Smad7 may provide a new therapeutic strategy for renal fibrosis.

Animals↗

PubMed2002

Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism.

Hyperuricemia is associated with hypertension and vascular disease, but whether this represents a causal relationship or an epiphenomenon remains unknown. We recently reported a model of mild hyperuricemia in rats that results in increased blood pressure and mild renal fibrosis. In this study, we examined the effect of hyperuricemia on the renal vasculature. Rats fed 2% oxonic acid and a low-salt diet for 7 wk developed mild hyperuricemia (1.8 vs. 1.4 mg/dl, P < 0.05), hypertension [147 vs. 127 mmHg systolic blood pressure (SBP), P < 0.05], and afferent arteriolar thickening, with a 35% increase in medial area (P < 0.05). Allopurinol or benziodarone prevented the hyperuricemia, hypertension, and arteriolopathy. Hydrochlorothiazide treatment did not prevent the hyperuricemia or arteriolopathy despite controlling blood pressure. In contrast, the arteriolopathy and hypertension were prevented by both enalapril and losartan. Uric acid also directly stimulated vascular smooth muscle cell proliferation in vitro, and this was partially inhibited by losartan. Thus hyperuricemia induces a renal arteriolopathy in rats that is blood pressure independent and involves the renin-angiotensin system.

Administration, Oral↗

PubMed2000

In vivo administration of a nuclear transcription factor-kappaB decoy suppresses experimental crescentic glomerulonephritis.

Glomerular expression of cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-alpha), together with leukocytic infiltration, are prominent features in crescentic glomerulonephritis. Because these cytokines are targets for nuclear transcription factor-kappaB (NF-kappaB), the use of NF-kappaB decoy oligodeoxynucleotide (ODN) treatment was evaluated in an experimental disease model. Crescentic glomerulonephritis was induced in primed Wistar rats by injection of sheep antiglomerular basement membrane serum. Thirty minutes after injection, rats were anesthetized and the left kidney was perfused with NF-kappaB decoy ODN or scrambled ODN control mixed with a virus-liposome complex, and then killed 7 d later. Animals given the scrambled control ODN developed severe glomerulonephritis by day 7 with heavy proteinuria, glomerular crescents and interstitial lesions, marked leukocytic infiltration, and upregulated renal expression of cytokines (IL-1 and TNF-alpha) and adhesion molecules (intercellular adhesion molecule-1). In contrast, NF-kappaB decoy ODN treatment substantially inhibited the disease with a 50% reduction in proteinuria, a threefold reduction in histologic damage, a 50% reduction in leukocytic infiltration, and a 50 to 80% reduction in the renal expression of cytokines and leukocyte adhesion molecules. In conclusion, this study has demonstrated that NF-kappaB plays a key role in cytokine-mediated renal injury and that NF-kappaB decoy ODN treatment has clear therapeutic potential in rapidly progressive glomerulonephritis.

Animals↗