Rheumatoid factor and immunoglobulin M mark hepatitis C-associated mixed cryoglobulinaemia: an 8-year prospective study.

OBJECTIVES: The prevalence and factors of hepatitis C virus (HCV) -associated mixed cryoglobulinaemia in Asia remain elusive, and we aimed to investigate these topics. METHODS: An 8-year prospective cohort study was conducted in 678 consecutive Taiwanese individuals with chronic HCV infection (438 completed an anti-HCV therapy course). RESULTS: Of 678 individuals, 437 (64.5%) had mixed cryoglobulinaemia and 20 (2.9%) had mixed cryoglobulinaemic syndrome. At baseline, IgM (cut-off >122 mg/dL), triglycerides and IgG levels, and HCV genotype 3 were independently associated with mixed cryoglobulinaemia. Rheumatoid factor (RF) levels were associated with mixed cryoglobulinaemic syndrome (cut-off >12.2 IU/mL). At 24 weeks post-therapy, the 362 individuals with a sustained virological response (SVR) had higher cured (106/362 (29.3%) versus 10/76 (13.2%), p = 0.003) and lower persistent (100/362 (27.6%) versus 33/76 (43.4%), p = 0.003) mixed cryoglobulinaemia rates than non-SVR patients. Among SVR patients, compared with baseline levels, RF, IgG and IgM levels decreased, except in individuals with new mixed cryoglobulinaemia. Pre-therapy IgM levels were associated with 24-week post-therapy new (95% CI of OR 1.002-1.023) and persistent (95% CI of OR 1.004-1.015) mixed cryoglobulinaemia in SVR patients. After up to 8 years, 24-week post-therapy IgM levels were associated with mixed cryoglobulinaemia in SVR patients (9/51; 17.64%; 95% CI of HR 1.004-1.011). Among 17 SVR patients with pre-therapy mixed cryoglobulinaemic syndrome, 5 (29.4%) had long-term mixed cryoglobulinaemia and 4 (23.5%) had mixed cryoglobulinaemic syndrome. CONCLUSIONS: Over 60% of chronic HCV-infected individuals had mixed cryoglobulinaemia, and 17.64% of SVR patients had mixed cryoglobulinaemia 8 years post-therapy. Pre-therapy RF and IgM levels marked HCV-associated mixed cryoglobulinaemic syndrome and mixed cryoglobulinaemia, respectively.

Interaction between hepatic membrane type 1 matrix metalloproteinase and acireductone dioxygenase 1 regulates hepatitis C virus infection.

Membrane type 1 matrix metalloproteinase (MT1-MMP) binds to and regulates the function of tetraspanin-enriched microdomains. It also physically interacts with claudin-1 and acireductone dioxygenase 1 (ADI1), both associated with hepatitis C virus (HCV) cell entry. Here, we examined hepatic expression of MT1-MMP, ADI1 and claudin-1 as well as their physical interaction in relation to serum or intrahepatic HCV-RNA levels. A total of 104 liver biopsies obtained from chronic hepatitis C patients and 84 liver tissues obtained from noncancerous parts of surgically removed HCV-related hepatocellular carcinoma were analysed. Positive cytoplasmic ADI1 in liver biopsies was associated with higher serum HCV-RNA levels (P = 0.009). Positive MT1-MMP and ADI1 interaction assessed by co-immunoprecipitation was associated with lower tissue HCV-RNA levels (P = 0.009). Hepatic HCV-RNA levels were positively associated with ADI1 levels in the MT1-MMP and ADI1 co-immunoprecipitates (P = 0.030). Overexpression of MT1-MMP in Huh7.5 cells suppressed cell entry of HCV pseudoparticles as well as HCVcc infection. The suppression effect could be reversed by co-expression of ADI1 in a dose-dependent manner. In summary, clinical and cell-based experiments suggested that physical interaction between MT1-MMP and ADI1 led to suppression of HCV infection. This inhibitory effect could be reversed by ADI1 overexpression.

Systematic review: cessation of long-term nucleos(t)ide analogue therapy in patients with hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND: It has been debated whether finite nucleos(t)ide analogue therapy is feasible in HBeAg-negative chronic hepatitis B. AIM: To review this issue systematically. METHODS: Using text terms HBsAg and various nucleos(t)ide analogues, PubMed was searched between 1995 and 2014 to find studies on therapy >6 months in adult HBeAg-negative chronic hepatitis B patients with off-therapy follow-up >6 months. RESULTS: Twenty-two studies with a total of 1732 patients were identified and included. The median duration of therapy, consolidation therapy and off-therapy follow-up ranged from 6 months to 8 years, 4 to 96 weeks and 6 to 80 months respectively. Patients were monitored with serum ALT and HBV DNA monthly in the first 1-3 months and every 3-6 months afterwards in most studies. The 1-year off-therapy 'virological relapse' (HBV DNA >2000 IU/mL) and 'clinical relapse' (HBV DNA > 2000 IU/mL + ALT elevation) occurred in <70% and <50% of the patients, respectively, and <40% of the patients received re-treatment. These rates were higher in patients with shorter treatment, shorter consolidation therapy and those treated with less potent nucleos(t)ide analogues. Off-therapy severe flares were rare and hepatic decompensation was reported in only one patient with cirrhosis. Biochemical relapse reflecting enhanced immune-mediated hepatocyte killing may lead to a higher chance for off-therapy HBsAg seroclearance and be possibly desirable. CONCLUSION: With an appropriate stopping rule and a proper off-therapy monitoring plan, cessation of long-term nucleos(t)ide analogue therapy prior to HBsAg seroclearance in HBeAg-negative chronic hepatitis B is a feasible alternative to indefinite treatment.

Hepatic expression of MxA and OAS1 in an ex vivo liver slice assay independently predicts treatment outcomes in chronic hepatitis C.

Antiviral effect of interferon is mediated by the expression of interferon-stimulated genes (ISGs). However, because of the difficulty in obtaining paired liver biopsies before and after interferon treatment, the key ISGs expressed in human hepatocytes and responsible for interferon-based antiviral activities in chronic hepatitis C remain illusive. Prior to a standard course of peginterferon plus ribavirin therapy, 104 patients underwent a liver biopsy. A small piece of the liver biopsy sample from each patient was submitted for ex vivo tissue culture in the presence or absence of interferon. Hepatic expression of 8 ISGs was detected by RT-PCR. The ISG expression patterns and clinicopathological variables were correlated with subsequent treatment outcomes. Multivariate logistic regression analysis showed that hepatic MxA expression (P = 0.008) and leucocyte count (P = 0.040) independently predicted the end of therapeutic virological response, while hepatic OAS1 expression (P = 0.003), genotype 1 (P = 0.002), HCV-RNA level (P = 0.007), AST/ALT ratio (P = 0.004) and leucocyte count (P = 0.034) independently predicted the sustained virological response. Immunohistochemistry analysis showed that interferon-induced OAS1 expression localized to the hepatocytes. In conclusion, hepatic MxA and OAS1 expression predicted, respectively, the end of therapeutic and sustained virological responses in interferon-based treatment of chronic hepatitis C.

Contact behavior of focused ion beam deposited Pt on p-type Si nanowires.

Pt contact on p-Si nanowires (NWs) using Ga-ion-induced deposition by a focused ion beam was formed with a specific contact resistance (rho(c)) of 1.54 x 10(-6) Omega cm(2). Ohmic behavior is caused by Ga-ion-induced amorphization of Si NWs underneath the Pt contact. A very low Schottky barrier height associated with interface states raised from Pt-amorphized Si junction and with an image force induced by the applied bias can be implemented to elucidate ultralow rho(c). The value of rho(c) lower than that of any known contact to Si NWs demonstrates a practical method for integrating NWs in devices and circuits.

A liver slice culture-based ex vivo assay to predict the outcome of antiviral therapy for chronic hepatitis C.

A liver slice culture-based, ex vivo drug suppression assay was developed as a pre-therapeutic predictor for the outcome of antiviral therapy. To investigate its clinical application, 106 consecutive patients with chronic hepatitis C virus (HCV) infection were evaluated. Ex vivo drug suppression assay was performed before administrating a standard course of peginterferon plus ribavirin combination therapy. Stepwise logistic regression model was used to estimate sustained virological response (SVR) on the presence of various clinicopathological parameters. Suppression of HCV replication in the ex vivo assay was present in 32 patients, 29 (90.6%) of whom achieved SVR. Stepwise logistic regression analysis indicated that the presence of interferon suppression effect in the ex vivo assay (odds ratio [OR], 5.552; 95% confidence interval [CI], 1.114-27.673; P = 0.036), genotype 1 (OR; 0.045, 95% CI, 0.008-0.259; P = 0.001), HCV-RNA level (OR, 0.739; 95% CI, 0.617-0.885; P = 0.001), the presence of fatty metamorphosis (OR, 0.205; 95% CI, 0.053-0.793; P = 0.022), and albumin (OR, 9.687; 95% CI, 2.237-41.940; P = 0.002) were independent determinants of SVR. Categorical analysis revealed that 17 of 17 (100%) patients with genotype non-1 and positive ex vivo suppression test achieved SVR, while 20 of 40 (50%) with genotype 1 and negative ex vivo suppression test achieved SVR. In conclusion, the ex vivo drug suppression assay may serve as an independent pre-therapeutic predictor for the SVR in interferon-based antiviral therapy.

Expression of hepatitis B virus nuclear core antigen in young cirrhotic patients is associated with an unfavourable long-term outcome.

The aim of this case control study was to investigate the clinical significance of hepatitis B virus nuclear core antigen (HBcAg) in young cirrhotic patients. Fifteen cirrhotic patients with nuclear HBcAg in the liver biopsies were included. Their clinicopathological parameters as well as the core gene sequences were compared with those of a sex- and age-matched (1 to 2) control group. The mean follow-up periods were 124 +/- 80 and 102 +/- 43 months, respectively. Expression of nuclear HBcAg in cirrhotic liver was significantly associated with higher aspartate aminotransferase levels (P = 0.001), alanine aminotransferase levels (P < 0.001), and alpha-fetoprotein levels (P = 0.002), as well as a shorter duration to develop hepatocellular carcinoma or liver decompensation (Kaplan-Meier method, P = 0.044). Sequence analysis revealed mutations on the nuclear localization signal (NLS) of core protein in five cirrhotic patients with nuclear HBcAg (Q171K in four and Q179K in one patients). Site-directed mutagenesis experiments demonstrated that both the Q171K and Q179K mutation enhanced nuclear localization of the core protein. In conclusion, expression of nuclear HBcAg in young cirrhotic patients was associated with more severe hepatitis activities as well as an unfavourable long-term outcome. Mutations on the NLS of core protein were selected in some patients with nuclear HBcAg.

Nuclear HBcAg and histology activity index as independent predictors of the expression of singly spliced HBV-RNA.

Although hepatitis B virus (HBV) RNA splicing has been reported by many researchers, the clinical significance of this event remains illusive. The present study was designed to investigate the clinical roles of singly spliced HBV-RNA. Liver biopsy tissues obtained from 32 consecutive patients were subjected to reverse transcriptase-polymerase chain reaction for the detection of singly spliced and unspliced HBV-RNA. Stepwise linear regression model was used to estimate the ratio of singly spliced to unspliced (S/US) HBV-RNA in the presence of the following variables: age, gender, aspartate aminotransferase, alanine aminotransferase, total bilirubin, alpha-foetoprotein, status of HBV e antigen (HBeAg), status of antibody to HBeAg, HBV-DNA, histology activity index (HAI), fibrotic score, grade of cytoplasmic HBV core antigen (HBcAg), grade of nuclear HBcAg, genotype, status of precore-stop-mutation, basal core promoter mutation, previous lamivudine therapy and superinfection by other hepatitis viruses. The results showed that HAI (beta = -0.2616; P = 0.011) and grade of nuclear HBcAg expression (beta = 0.5599; P =0.0067) were two independent predictors for the expression of singly spliced HBV-RNA. Further categorical analysis showed that patients with HAI score or=2 have significantly higher S/US ratios. In conclusion, nuclear HBcAg and HAI are two independent predictors for the expression of singly spliced HBV-RNA.

Identification of variations in the human phosphoinositide 3-kinase p110delta gene in children with primary B-cell immunodeficiency of unknown aetiology.

Our recent study demonstrated that defects in p110delta result in B-cell immunodeficiency that is very similar to that observed in BTK-deficient mice. We revealed that the p110delta fit the B-cell signal transduction complex and played a non-redundant role in the development and function of B cells. In humans, most children with primary B-cell immunodeficiency have mutations in the BTK, whereas a few have defects in the components of the B-cell signal transduction complex. But little is known about the genetic variation of p110delta in children with defects in B-cell immunodeficiency of unknown aetiology. Sixteen patients from 15 unrelated families and 112 normal controls underwent sequence analysis to identify genetic variations of the p110delta. Allele frequency in each group was also analysed and compared. We identified five single base-pair polymorphic nucleotide exchanges in both patient and control groups with similar allele frequencies, which did not contribute to the immunodeficiency. Three of them are novel (m.953A>G, m.1200C>T and m.1561A>G), and the m.953A>G and m.1561A>G nucleotide exchanges are non-synonymous (N253S and T456A, respectively). The novel m.1561A>G was in complete linkage disequilibrium with the known m.873A>G in our study of Taiwanese group. In addition, one novel single base-pair missense mutation, m.3256G>A (E1021K), was identified in one boy with typical clinical features of primary B-cell immunodeficiency and could not be found in either his family or the normal control population. By atomic structural analysis of the amino acid as well as the alignment comparison between species, it resulted in the replacement of the negative-charged amino acid E with the positive-charged amino acid K at codon 1021, located in the highly conservative and important catalytic functional domain. Our findings could shed light on further understanding the polymorphisms of p110delta in B-cell immunodeficiency and different populations. Moreover, the 3256G>A missense mutation raised the attention and warranted further extensive analysis to elucidate the role of p110delta in human immunodeficiency.

Cost effectiveness and delivery study for future HIV vaccines.

Research teams from five countries, Brazil, China, Kenya, Peru and Thailand, have initiated a policy-maker survey on vaccine delivery, cost studies for future HIV vaccination programmes, and associated simulation modeling exercises analysing the relative cost-effectiveness of potential HIV vaccination strategies. The survey assesses challenges and opportunities for future country-level HIV vaccination strategies, providing data on the vaccine characteristics (e.g. vaccine efficacies for susceptibility, infectiousness and disease progression) and vaccination programme strategies to be considered in the cost-effectiveness modeling analyses. The study will provide decision-makers with modeling data on vaccination policy considerations that will assist in developing country-level capacities for future HIV vaccine policy adoption and effective delivery systems, and will help delineate the long-term financial requirements for sustainable HIV vaccination programmes. The WHO-UNAIDS HIV Vaccine Initiative and the collaborating researchers welcome comments or questions from policy makers, health professionals and other stakeholders in the public and private sectors about this effort to help advance policy and capacity related to future potential HIV vaccines.

Signal transduction pathways of nitric oxide release in primary microglial culture challenged with gram-positive bacterial constituent, lipoteichoic acid.

Between one-third and one-half of all cases of sepsis are known to be caused by gram-positive microorganisms through the cell wall component, e.g. lipoteichoic acid (LTA). Gram-positive bacteria are also known to induce encephalomyelitis and meningeal inflammation, and enhance the production of nitric oxide (NO) via expression of inducible nitric oxide synthase (iNOS) in murine tissue macrophages. It remains to be explored if LTA could activate microglia considered to be resident brain macrophages. We report here that LTA derived from gram-positive bacteria (Staphylococcus aureus) significantly induces NO release and iNOS expression in primary microglia. LTA-induced NO accumulation was detected at 2 h in microglial culture and was significantly attenuated by pretreatment with anti-CD14, complement receptor type 3 (CR3) or scavenger receptor (SR) antibodies. LTA activated mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase, p38 MAPK or c-Jun N-terminal kinase in cultured microglia. LTA-elicited microglial NO production was also drastically suppressed by SB203580 (p38 MAPK inhibitor) or pyrrolidine dithiocarbamate (an inhibitor of nuclear factor kappaB), indicating that p38 MAPK and nuclear factor kappaB were involved in microglial NO release after LTA challenge. These results suggest that gram-positive bacterial product such as LTA can activate microglia to release NO via the signal transduction pathway involving multiple LTA receptors (e.g. CD14, CR3 or SR), p38 MAPK and nuclear factor kappaB. The in vivo study further confirmed that administered intracerebrally LTA induced considerable noticeable iNOS, phospho-IkappaB and phospho-p38 MAPK expression in microglia/macrophages.

Breaking new ground--are changes in immunization services needed for the introduction of future HIV/AIDS vaccines and other new vaccines targeted at adolescents?

A safe, effective and accessible preventive vaccine is our best long-term hope for the control of the HIV/AIDS pandemic. Once the first generation of HIV vaccines are developed, many questions remain unanswered regarding their administration. For instance, which vaccines should be given to whom at what age and how many doses? We argue that pre- and early-adolescents will be one of the main target groups for future HIV vaccines, that is, before the age of exposure to the virus. Historically, immunization has mainly focused on infants. Indeed, vaccines have only occasionally been systematically targeted at adolescents, even in industrialized countries. Delivering vaccines to pre-adolescents and adolescents in developing countries would, to a great extent, be a new challenge. But it is not just HIV/AIDS vaccines that are coming down the pipeline. Herpes simplex type2 (HSV-2) and human papillomavirus (HPV) vaccines are also among the exciting candidate vaccines that may be the agents of change needed to encourage even the poorest countries to develop strategies for reaching adolescents with vaccines and other health services in the coming decade. Together, they may also provide the impetus for changing the paradigm for how vaccines are administered. Not only will more antigens be included in national immunization schedules, but the age of target groups will range much more widely than at present, encompassing older children, adolescents and young adults. While presenting major difficulties for delivery, these new ingredients also offer stimulating opportunities to completely rethink how vaccines are presented, administered and delivered. We predict that even the poorest countries will be looking to developing integrated, sustainable strategies for reaching pre-adolescents and adolescents with vaccines in the coming decade.

Recent developments on the role of mitochondria in poly(ADP-ribose) polymerase inhibition.

Numerous pathophysiological disorders involve some element of oxidative stress and bioenergetic deficit. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been used recently as a promising new therapeutic strategy aimed at halting the bioenergetic decline associated with oxidative brain insults and other conditions. PARP-1 uses NAD+ as a substrate and is activated during stressful circumstances, mainly in the nucleus. PARP-1 inhibitors are well known for blocking the excessive consumption of NAD+, thereby preserving energy metabolism. But what is the role of mitochondria in this process? Recent investigations have begun to focus on whether mitochondrial function can also be preserved by PARP-1 inhibitors. This review will present some of the latest mechanistic evidence documenting the potential involvement of PARP-1 inhibitors in protecting mitochondrial function and preventing necrosis, apoptosis and mitochondrial calcium cycling.

Public health considerations for the use of a first generation HIV vaccine. Report from a WHO-UNAIDS-CDC consultation, Geneva, 20-21 November 2002.

SUMMARY: To accelerate the development and future availability of safe, effective and affordable HIV vaccines it is essential to address not only the associated biomedical obstacles, but also the logistic aspects that would guide the introduction and use of those vaccines. It is likely that initial vaccines may only be partially effective, and their public health use will have to be carefully considered. This report summarizes the discussions from a consultation held in Geneva (20-21 November 2002) organized by the World Health Organization (WHO), the joint United Nations Programme on HIV/AIDS (UNAIDS) and the US Centers for Disease Control and Prevention (CDC). The group identified a number of logistic issues that need to be addressed to accelerate the development and future availability of HIV vaccines, and made broad recommendations in four different areas: (a) Vaccine manufacturing and licensing; (b) vaccination acceptability and social marketing; (c) immunisation strategies and delivery; and (d) access and economic issues. The implementation of these recommendations will require the participation of multiple stakeholders in the public and private sector, in industrialized and developing countries. These actions will be essential to ensure widespread and rapid access to HIV vaccines globally, soon after their efficacy is demonstrated in clinical trials.

Parkinson's disease--redox mechanisms.

Parkinson's disease occurs in 1% of people over the age of 65 when about 60% of the dopaminergic neurons in the substantia nigra of the midbrain are lost. Dopaminergic neurons appear to die by a process of apoptosis that is induced by oxidative stress. Oxygen radicals abstract hydrogen from DNA forming DNA radicals that lead to DNA fragmentation, activation of DNA protective mechanisms, NAD depletion and apoptosis. Oxygen radicals can be formed in dopaminergic neurons by redox cycling of MPP+, the active metabolite of MPTP. This redox cycling mechanism involves the reduction of MPP+ by a number of enzymes, especially flavin containing enzymes, some of which are found in mitochondria. Tyrosine hydroxylase is present in all dopaminergic neurons and is responsible for the synthesis of dopamine. However, tyrosine hydroxylase can form oxygen radicals in a redox mechanism involving its cofactor, tetrahydrobiopterin. Dopamine may be oxidized by monoamine oxidase to form oxygen radicals and 3,4-dihydroxyphenylacetaldehyde. This aldehyde may be oxidized by aldehyde dehydrogenase with the formation of oxygen radicals and 3,4-dihydroxyphenylacetic acid. The redox mechanisms of oxygen radical formation by MPTP, tyrosine hydroxylase, monoamine oxidase and aldehyde dehydrogenase will be discussed. Possible clinical applications of these mechanisms will be briefly presented.

Brain oxidative stress--analytical chemistry and thermodynamics of glutathione and NADPH.

Oxidative stress occurs in the brain due to stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, trauma, aging and other conditions. Analysis of the effects of oxidative stress can involve quantitation of brain GSH, GSSG, NADPH and NADP. Reliable and rapid assays have been developed for these compounds and will be presented in detail. The assays have been used to analyze the effects of brain oxidative stress. Thermodynamic calculations can be performed to find the observed electrochemical potentials of the GSSG/GSH and the NADP/NADPH couples during oxidative stress. The biochemical consequences of these thermodynamic changes in the cell will be discussed as well as the defense mechanisms available to the cell to recover from oxidative stress.

Association between genetic polymorphism of tumor necrosis factor-alpha and risk of oral submucous fibrosis, a pre-cancerous condition of oral cancer.

Many cytokines have been thought to play important roles in the pathogenesis of oral submucous fibrosis (OSF), an areca nut chewing-specific pre-cancerous condition characterized by the deposition of collagen in oral submucosa. Tumor necrosis factor-alpha (TNF-alpha), situated in the class III region of human leukocyte antigen (HLA), is a mediator with multiple functions, including the regulation of inflammatory reaction and transcriptions of collagen and collagenase. In total, 809 male subjects were recruited for assessment of the association of OSF with a bi-allelic promoter-region (-308) polymorphism on the TNFA gene. The high production allele, TNF2, was significantly lower among OSF subjects (n = 166) than in areca-chewing controls (n = 284). This association was independent of oral cancer status. The multivariate-adjusted odds ratio for the TNFA 11 genotype was 2.6 (95% confidence interval = 1.4-4.9; p = 0.004). The finding may imply a multifunctional etiological factor of TNF-alpha in OSF pathogenesis.

Cyclical mechanical stretching increases the expression of vascular endothelial growth factor in rat vascular smooth muscle cells.

BACKGROUND AND PURPOSE: Mechanical forces have profound effects on vascular smooth muscle cells (VSMCs). The mechanism by which mechanical stimuli regulate vascular endothelial growth factor (VEGF) expression and regulation has yet to be elucidated. We investigated the effect of cyclical mechanical stretching on regulation of the VEGF gene in VSMCs. MATERIALS AND METHODS: Cultured rat VSMCs grown on a flexible membrane base were stretched by applying a vacuum at 60 cycles/minute. VEGF concentration in the cultured media was determined by enzyme-linked immunoassay. VEGF gene expression was determined by Western blot and Northern blot. The location of VEGF in the VSMC was studied immunohistochemically. Chimeric constructs of the VEGF promoter were deleted and the promoter activity was determined by luciferase activity. RESULTS: VEGF concentration increased by 21 to 32% as early as 10 minutes after stretching and remained at this level for up to 12 hours. The concentration of VEGF reached a maximum of 2.8-fold over that in control cells by 2 hours after stretching and declined slightly thereafter. The amount of VEGF mRNA in stretched cells increased as early as 1 hour after stretching, reached a maximum of 3.2-fold over the amount in control cells by 2 hours, and remained at this level for up to 6 hours after stretching. Immunohistochemical study confirmed increased VEGF expression in VSMCs after stretching. Stretched cells transfected with a Sac-Nhe fragment showed only 46% of the luciferase activity of unstretched control cells. However, stretched cells transfected with chimeric plasmids containing a Spe-Nhe fragment showed 2.8-fold luciferase activity over that in control cells. CONCLUSIONS: Cyclical mechanical stretching upregulates expression of the VEGF gene in VSMCs at the transcription level. The VEGF 5'-flanking region contains a negative stretch-response element located in the 0.4-kb Sac-Pst fragment and a positive stretch-response element located in the 0.6-kb Spe-Sac fragment.