HIV pre-exposure prophylaxis during the SARS-CoV-2 pandemic: Results from a prospective observational study in Germany.

Aims: Since 2017, HIV pre-exposure prophylaxis (PrEP) care has been provided through an intersectoral collaboration at WIR (Walk-in-Ruhr, Center for Sexual Health and Medicine, Bochum, Germany). The aim of this study was to establish possible impact of COVID-restrictions on the sexual behavior of PrEP users in North Rhine-Westphalia. Methods: The current PrEP study collected data of individuals using PrEP, their sexual behavior and sexually transmitted infections (STIs) before (each quarter of year 2018) and during the COVID-19 pandemic (each quarter of year 2020). Results: During the first lockdown in Germany from mid-March until May 2020, PrEP-care appointments at WIR were postponed or canceled. Almost a third of PrEP users had discontinued their PrEP intake in the 2nd quarter of 2020 due to alteration of their sexual behavior. The number of sexual partners decreased from a median of 14 partners in the previous 6 months in 1st quarter of 2020, to 7 partners in 4th quarter of 2020. Despite such a significant reduction in partner number during the pandemic in comparison to the pre-pandemic period, a steady rate of STIs was observed among PrEP users in 2020. Conclusion: The SARS-CoV-2-pandemic has impacted PrEP-using MSM in North Rhine-Westphalia with respect to their PrEP intake regimen and sexual behavior in 2020. Our study revealed a steady rate of STI among PrEP users even during the pandemic, thus highlighting the importance of ensuring appropriate HIV/STI prevention services in times of crisis.

Influence of dietary supplementation of short-chain fatty acid sodium propionate in people living with HIV (PLHIV).

BACKGROUND: Non-AIDS-associated chronic diseases in HIV+ patients have been on the rise since the advent of antiretroviral therapy. Especially cardiovascular diseases and disruption in the gastrointestinal tract have limited health-related quality of life (QoL). Several of those complications have been associated with chronic systemic inflammation. Short-chain fatty acids (SCFA), with propionate as one of the major compounds, have been described as an important link between gut microbiota and the immune system, defining the pro- and the anti-inflammatory milieu through direct and indirect regulation of T-cell homeostasis. The effects of dietary supplementation of sodium propionate (SP) in people living with HIV (PLHIV) have not yet been investigated prior to this study. OBJECTIVES: To investigate the impact of SP uptake among PLHIV and its relevance to improve QoL, the study aimed to investigate metabolic, immunological, microbiome and patient-reported QoL-related changes post-SP supplementation with follow-up. METHODS: A prospective, non-randomized, controlled, monocentric interventional study was conducted in WIR, Center for Sexual Health and Medicine, in Bochum, Germany. 32 HIV+ patients with unaltered ART-regimen in the last three months were included. Participants were given SP for a duration of 12 weeks in the form of daily oral supplementation and were additionally followed-up for another 12 weeks. RESULTS: The supplementation of SP was well tolerated. We found an improvement in lipid profiles and long-term blood glucose levels. A decrease in pro-inflammatory cytokines and a depletion of effector T cells was observed. Regulatory T cells and IL-10 decreased. Furthermore, changes in taxonomic composition of the microbiome during follow-up were observed and improvement of items of self-reported life-quality assessment. CONCLUSION: Taken together, the beneficial impact of SP in PLHIV reflects its potential in improving metabolic parameters and modulating pro-inflammatory immune responses. Thus, possibly reducing the risk of cardiovascular disorders and facilitating long-term improvement of the gut flora.

[Diagnostic workup and treatment of sexually transmitted infections]. / Diagnostik und Therapie von sexuell übertragbaren Infektionen.

There are a number of reasons to exclude sexual transmitted infections (STI). In addition to testing patients presenting with symptoms like discharge, burning when urinating or genitals ulcers, risk-adapted STI tests should be offered. Medical history taking should include sexual orientation and practices, symptoms also of sex partners and symptom-free intervals, previous STI and other medical conditions (especially allergies against antibiotics), previous treatment, vaccination status, contraceptive methods and condom use. Clinical examination depends on anatomy, sexual practices and symptoms. It should always include the inspection of the genital and anal region, the throat and the skin. The goal is to also diagnose oral and anal STI and to provide guideline-based treatment including monitoring of outcome.

Sexual behaviour, STI knowledge and Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) prevalence in an asymptomatic cohort in Ruhr-area, Germany: PreYoungGo study.

BACKGROUND AND OBJECTIVES: STIs present a significant threat to individual and public health, disproportionately affecting youth. The study aimed to evaluate (a) the prevalence of asymptomatic Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections among youth using a rapid assay platform, (b) the participants' sexual behaviour and STI knowledge, (c) the utility of the rapid assay in reducing diagnosis-to-treatment time. METHODS: In this study, 272 subjects (14-31 years) were included between 12/2016 and 7/2018. A questionnaire was used to collect sociodemographic data, sexual behaviour and STI knowledge. Prevalence of CT and NG infections were tested from oral, vaginal and anal swabs for women and oral, anal swabs and urine for men, using the Cepheid Xpert® CT/NG assay. Time intervals between (i) test to the time the patient were informed of the result (turn around time - TAT) and (ii) test to therapy initiation was documented. RESULTS: Of the 272 subjects (48.9% female, 48.9% male, undisclosed 2.2%), 56.6 % reported university education. 46.6% were men who have sex with men (MSM), and 47.4% of women and 63.1% of men had anal intercourse. 59.9% had previously been tested for HIV, while only 39.7% had for CT, 20.6% for NG. Among these asymptomatic youth 7.7% were positive for CT and 5.5% for NG. The localization of CT were 3.7% genital, 5.5% anal and 2.2% oral, while the corresponding localization of NG were 0.4%, 2.9%, 4.4% respectively. 91.8% of the participants were informed of a positive result within 24 h with a median TAT of 03:09 h. 73.3% initiated therapy within 24 h, with a median time from testing to therapy initiation being 06:50 h. CONCLUSION: Asypmtomatic CT and NG infections are common and often not tested in persons at risk. The Cepheid Xpert® CT/NG assay is an effective strategy as it reduces STI diagnosis-to-treatment time to less than a day.

Age specific evaluation of sexual behavior, STI knowledge and infection among asymptomatic adolescents and young adults.

OBJECTIVES: Adolescents and young adults account for 50% of new infections with sexually transmitted infection (STI), while representing only 25% of the sexually active population. An evaluation of sexual behavior, STI knowledge and infection among youth of different age groups is relevant to formulate effective sexual health strategies. METHODS: In this monocentric, open, prospective cross-sectional study, 268 asymptomatic subjects (14-30 years) were partitioned into three cohorts I: 14-19 (n=42), II: 20-25 (n=147) and III: 25-30 (n=79) years. A comparative analysis of their sexual behavior and STI knowledge was performed using a 21-item questionnaire at the WIR-Center for Sexual Health and Medicine. Prevalence of chlamydia and gonococcal infections were tested, using the Cepheid Xpert® CT/NG assay. RESULTS: The three age-specific cohorts showed statistically significant differences in their relationship status, their sexual contacts and the number of life-time sexual partners. Independent of age, men who have sex with men had greater number of sexual partners across all age groups. Although >95% of participants were aware of HIV in all age groups, the corresponding awareness of non-HIV STI was consistently lower in cohort I. The awareness of trichomonas, mycoplasma and candida in particular was <50% across all agedemographies. Chlamydia screening remains poor despite current chlamydia infection among cohort I, II & III being 7.1%, 5.4%, & 11.4% respectively. CONCLUSION: Our study demonstrates a demographic divide in the knowledge on most frequent STI apart from HIV. Current education, screening and vaccination programs for STI among the younger demographic should be improved.

[Profile of Kaposi sarcoma patients in the competence network HIV/AIDS]. / Profil der Kaposi-Sarkom-Patienten im Kompetenznetz HIV/AIDS.

BACKGROUND: Kaposi's sarcoma (KS) represents the most common AIDS-defining neoplasm. Only very few studies regarding the course and treatment of human immunodeficiency virus (HIV)-associated KS have been carried out in Germany. OBJECTIVE: In this study the course of HIV-associated KS was observed in patients from the cohort database of the competence network for HIV/AIDS. MATERIAL AND METHODS: Data from HIV-associated KS patients from 9 German core centers from 1987 to 2011 were retrospectively collected. Kaplan-Meier curves for the recurrence and survival probability were calculated. RESULTS: In 222 patients KS was diagnosed at a median age of 38.5 ± 10.1 years. Men were almost exclusively affected (97.7%). The HIV viral load at the time of diagnosis was in 7.4% <50 copies/ml. Of the patients 55.5% developed KS with a CD4 cell count of <200 cells/µl and 9.5% with >500 cells/µl. In 68 patients KS therapy consisted exclusively of the optimization or initiation of antiretroviral therapy (ART). In addition, 71 patients were treated with pegylated liposomal doxorubicin. During the median follow-up period of 8.9 ± 4.9 years, 80.2% of the patients were free of KS recurrence. Survival rates after 5 and 10 years were 96.8% and 91.3%, respectively. CONCLUSION: Even with a good immune status HIV-associated KS occurred. An effective ART was the most important mainstay of therapy. With appropriate therapy, HIV-positive patients with KS showed a good survival rate.

MicroRNA-155 is a biomarker of T-cell activation and immune dysfunction in HIV-1-infected patients.

OBJECTIVES: MicroRNA-155 (miR-155) regulates T-cell differentiation and activation. It has also been associated with HIV infection. However, it remains unclear whether miR-155 is related to the T-cell response in HIV-infected individuals (e.g. T-cell activation and exhaustion). METHODS: We performed a cross-sectional study involving 121 HIV-1-infected patients on highly active antiretroviral therapy (HAART) and 43 HAART-naïve patients. MiR-155 levels in the peripheral blood were determined by quantitative reverse transcription-polymerase chain reaction (PCR). T-cell immune activation, exhaustion, and homeostasis were measured by determining the expression of CD38, programmed death 1 (PD-1) and CD127 via flow cytometry. RESULTS: The levels of miR-155 in total peripheral blood mononuclear cells, CD4 T cells and CD8 T cells from HIV-1-infected patients were increased (P < 0.01). Nonresponders and HAART-naïve patients also exhibited a higher percentage of CD8+ CD38+ T cells and a lower percentage of CD4+ CD127+ and CD8+ CD127+ T cells (P < 0.05). We also found higher levels of PD-1 expression on the CD4+ and CD8+ T cells of HIV-1-infected patients (P < 0.05). CONCLUSIONS: Our findings suggest that miR-155 levels in the peripheral blood of HIV-1-infected patients are increased and associated with T-cell activation. Therefore, miR-155 is a potential biomarker of the immune response following HIV-1 infection.

[Local health study : Outreach medical services for female sex workers in Bochum]. / Local Health Study : Aufsuchende medizinische Angebote für Sexarbeiterinnen in Bochum.

BACKGROUND: Inadequate access to prevention and medical treatment for female sex workers (SW) represents a challenge for the German health system. Accessibility and care for SW in Bochum (Germany) through a cooperation between the Interdisciplinary Immunology Outpatient Clinic, Center for Sexual Health and Medicine of St. Josef's Hospital, the Bochum health department and the Madonna e.V. was the focus of this work. PATIENTS AND METHODS: Medical outreach services were provided for the diagnosis of sexually transmitted infections (STI) in SW in brothels in Bochum between August 2013 and January 2014. After clarification and verbal consent from the SW, free HIV, syphilis, chlamydia, gonorrhea and trichomoniasis tests were offered and carried out using pseudonyms for the SW. RESULTS: A total of 112 SW were reached (up to 55.4 % within the framework of the STI Outreach Study). Of the SW, 94.6 % had an immigrant background. The majority (61.3 %) of SW were between 20 und 29 years old. Only 19.0 % of the collective had health insurance. The following STIs were diagnosed: 12.5 % chlamydia, 6.2 % syphilis, 3.6 % gonorrhea, 3.6 % trichomoniasis, and 0.9 % HIV. These results were compared with results from STI studies in SW in Germany. Treatment was performed in accordance with the standards of the German STI Society. CONCLUSION: The offer improved the accessibility and the utilization of medical services by SW in Bochum. A further improvement of services is urgently needed.

Viral load strategy: impact on risk behaviour and serocommunication of men who have sex with men in specialized care.

BACKGROUND: Incidence and prevalence of HIV are continuously high in German men, who have sex with men (MSM). Different transmission risk minimizing strategies have been observed. The viral load strategy rates patients unlikely to be sexually infectious if their viral load under effective therapy is stably suppressed during 6 months and no other sexually transmitted infections are present. OBJECTIVES: We aim to objectify the current popularity of the viral load strategy, the adherence to basic conditions and its impact on risk behaviour and serocommunication. Until now, no data on a German sample of HIV-positive MSM in regular specialized outpatient care are available. METHODS: Cross-sectional study with group comparisons between user group and non-user-group of the viral load strategy. Self-report questionnaires were conducted with 269 sexually active German HIV+MSM under effective treatment in specialized outpatient care. Structured interviews gathered additional information about approach to and realization of definite action levels concerning sexual risk behaviour and transmission risk minimizing strategies. RESULTS: Twenty-seven of 269 participants (10%) affirmed knowledge of having an undetectable viral load and stated this to be criteria for unprotected sexual behaviour. This subgroup reported more unprotected insertive (P = 0.018) and receptive anal intercourse (P = 0.042), more anonymous sex partners (P = 0.008) and less consistent safer sex. Analysing serocommunication, less addressing HIV/AIDS in general (P = 0.043) and less disclosing to sex partners (P = 0.023) was found, especially in anonymous settings. Differentiating serocommunication characteristics, a focus on seroguessing was depicted. CONCLUSIONS: The user group of the viral load strategy is small. But a less frequent, more reactive and assumptive serocommunication leads to an imprecise information exchange paired with higher frequency of risky behaviour, especially in anonymous settings, where frank serocommunication is often avoided. The targeted group of the viral load strategy diverges greatly from the user group.

The male ScreenING Study: prevalence of HPV-related genital and anal lesions in an urban cohort of HIV-positive men in Germany.

BACKGROUND: Human papillomaviruses (HPV) induce condylomata, anogenital cancers and their precursor lesions as anal or penile intraepithelial neoplasia (AIN/PIN). HIV-positive individuals have an increased risk for the development of anogenital HPV-induced lesions. OBJECTIVE: Estimation of the prevalence of HPV-related anogenital benign and malignant lesions in HIV-infected men attending a screening programme. METHODS: Four hundred HIV-positive men [98% men who have sex with men (MSM)] were enrolled in this prospective study from 2008 to 2011. All patients received an inspection of the anogenital region, digital rectal examination, high-resolution anoscopy (HRA), anal cytology, anal/penile histology if required, and HPV-typing of anal and penile swabs. RESULTS: At baseline, 75% (n = 302) of the men had abnormal anal cytological/histological results. 41% presented with low-grade (n = 164), 24% with high-grade anal dysplasia (n = 95) and two men with invasive anal cancer. 2.3% had PIN (n = 9) and one patient had penile cancer at baseline. Throughout the study period, 75% had anal dysplasia (low-grade n = 177, high-grade n = 125), 3.3% (n = 13) had PIN and two further patients developed anal cancer. Within the study period, 52.8% (n = 211) had condylomata (49% anal, 15% penile, 11% anal plus penile condylomata). At baseline, 88.5% of anal and 39.3% of penile swabs were HPV-DNA positive, and 77.8% of anal and 26.5% of penile swabs carried high-risk HPV-types. HPV16 was the most frequent HPV-type. CONCLUSION: HIV-positive MSM have a high risk for HPV-induced condylomata, (pre)malignant anogenital lesions and anogenital cancers. Screening for HPV-induced dysplasia is crucial to avoid progression to invasive carcinomas. Additionally, HPV-vaccination recommendations should be extended to high-risk populations.

Study of T cell subsets and IL-7 protein expression in HIV-1-infected patients after 7 years HAART.

OBJECTIVE: To study the changes in T cell subsets and IL-7 in HIV-1-infected patients after seven years of highly active antiretroviral therapy (HAART). METHODS: Seventy-five individuals were included in this study (25 with effective HAART, 18 with ineffective HAART, 17 untreated HIV+ patients, and 15 volunteers in the HIV negative control group). The counts of CD4+, CD8+, CD8/CD38+, and CD8/HLADR+ T cells as well as the IL-7 protein expression was measured at 5 time points during a period of seven years in patients starting HAART (baseline) and in the HIV negative control group. The expression of CD127 on CD3+ T cells was measured by flow cytometry at a single time point (after 7 years) in patients with HAART and was compared with untreated HIV+ patients and the HIV negative control group. RESULTS: At baseline CD4+ T cell counts of HIV-1-infected patients were lower than that in the control group (p < 0.01), whereas the CD8+, CD8/HLADR+ and CD8/CD38+ T cell counts were higher than those in the control group (p < 0.01). After seven years of effective HAART, the CD4+ T cell counts had increased and the CD8+ T cell count had decreased, although not to the normal levels (p < 0.05). Both the CD8/HLADR+ and CD8/CD38+ T cell counts had gradually approached those of the control group (p > 0.05). In the ineffective HAART group, the CD8/CD38+ T cell count had not decreased significantly, and CD8/HLADR+ T cell count gradually decreased. Before treatment, IL-7 serum levels of patients were significantly higher than that in the control group (p < 0.01). After seven years of effective HAART, IL-7 levels had gradually decreased, but were still higher than in the control group (p < 0.01). The CD127 expression on CD3+ CD8+ T cells in effective HAART patients was higher than in untreated HIV+ patients (p < 0.05), but was lower than that in the control group (p < 0.05). CD127 expression on CD3+ CD4+ T cells was not significantly different among the control group, untreated HIV+ patients and effective HAART group. CONCLUSION: After seven years of effective HAART, the quantity and capacity of T cell subsets and IL-7 in HIV-1-infected patients had been partially restored, and the abnormal immune activation has significantly diminished.

Epidemiological composition, clinical and treatment characteristics of the patient cohort of the German Competence Network for HIV/AIDS.

OBJECTIVE: As its central basis for research, the Competence Network for HIV/AIDS (KompNet) established a nationwide cohort study on HIV-positive patients being in medical care in Germany. In this paper, we describe the epidemiological composition, and clinical as well as treatment characteristics of the KompNet cohort over time. METHODS: The KompNet cohort is an open, retrospective and prospective, multi-center, disease-specific and nationwide cohort study that started gathering data in June 2004. Semiannually, follow up visits of the patients are documented, covering a wide range of clinical and sociodemographic data. At enrollment and three years afterwards, an EDTA-sample is taken; a serum-sample is taken at every follow up. RESULTS: As of 20.10.2008, a total of 15,541 patients were enrolled by 44 documenting sites. In September 2007, the cohort size was reduced to ten outpatient clinics and fifteen private practitioners, covering a total of 9,410 patients. The documentation of these patients comprised 24,117 years of follow up-time since enrollment (mean: 2.6 years), 62,862 person years inclusive data documented retrospectively on course of HIV-infection and antiretroviral therapy (ART, mean: 6.7 years). Due to the short period of recruitment till now, rates of death (0.3%-0.8%) and losses to follow up (1.1%-5.5%) were low. 84.9% of patients were men. Main risk of transmission was sex between men (MSM: 62.9%). Mean age was 45 years. About two third of patients were classified as CDC-stage B or C. Therapy regimens of currently treated patients complied with recent guidelines. Trends of mean CD4 cell count/microl regarding the initial therapy and concerning the population under treatment reflected the developments and the changing standards of antiretroviral therapy over time. CONCLUSION: The KompNet cohort covers about a quarter of all patients estimated as being under treatment in Germany. Its composition can be accounted approximately representative for the situation of clinical care and treatment in the scope of HIV/AIDS in Germany. Therefore, it is an important instrument for measuring the course of HIV/AIDS, the reality of use of antiretroviral therapy and its clinical and psychosocial outcomes in Germany.

The Patient Cohort of the German Competence Network for HIV/AIDS (KompNet): a profile.

OBJECTIVE: In this paper, we describe the main objectives, the study design and the onset of the patient cohort of the German Competence Network for HIV/AIDS (KompNet) (www.kompetenznetz-hiv.de). Furthermore, we depict sociodemographic and clinical baseline characteristics and an estimation of the coverage and representativity as to the composition of persons living with HIV/AIDS (PLWHA) in Germany. METHODS: The KompNet cohort is an open, retrospective and prospective, multicenter, disease-specific and nationwide cohort study that started gathering data in June 2004. Semi-annually, follow up visits of the patients are documented, covering clinical and sociodemographic data. At enrolment and three years afterwards, an EDTA-sample is taken; a serum-sample is taken at every follow up visit. RESULTS: As of 14.9.2008, a total of 15,541 patients were enrolled by 44 documenting sites. In September 2007, the cohort size was reduced to 10 outpatient clinics and fifteen private practitioners, covering a total of 9,410 patients. The documentation of these patients comprises 24,117 years of follow up-time since enrolment (mean: 2.6 years), 62,862 person years inclusive data documented retrospectively on course of HIV-infection and combined antiretroviral therapy (cART, mean: 6.7 years). 1,008 patients (10.7%) were lost to follow up and 175 (1.9%) died since enrolment. 84.9% of patients were men. Main risks of transmission were sex between men (MSM: 62.9%), heterosexual contacts (18.4%), intravenous drug use (IVDU: 7.0%) and origin from a high prevalence country (HPL: 5.2%). Mean age was 45 years. CONCLUSION: The KompNet cohort covers about a quarter of all patients being under treatment in Germany. The composition of the cohort represents well the most important risks of transmission in Germany. The cohort contains a high proportion of patients being older than 49 years (28.1%). On basis of its comprehensive database and its biomaterials banks, the KompNet cohort serves as an important instrument to monitor and analyse the effects of combined antiretroviral therapy (cART) in Germany, interdigidating basis, clinical and psychosocial research in view to translational research.

Lovastatin blocks basic fibroblast growth factor-induced mitogen-activated protein kinase signaling in coronary smooth muscle cells via phosphatase inhibition.

We have recently reported that the activation of mitogen-activated protein kinase (MAPK) through specific protein kinase C (PKC) isoforms is required for basic fibroblast growth factor (bFGF)-induced proliferation of coronary smooth muscle cells (cSMC). In this study, we investigated the effects of the 3hydroxy-3-methyl glutaryl coenzyme A (HMG CoA) reductase inhibitor lovastatin on bFGF-induced signal transduction in cSMC. The present study shows that lovastatin inhibits bFGF-stimulated DNA synthesis in cSMC, and that this inhibition is reversed by mevalonate (50 micromol/l) and by geranylgeranyl-pyrophosphate (1-5 micromol/l). Although lovastatin prevented Ras farnesylation the amount of bFGF-stimulated MAPK phosphorylation decreased only partially after lovastatin treatment. In addition, lovastatin pretreatment resulted in a sustained phosphorylation of MAPK. We observed a dose-dependent lovastatin-dependent increase in PKC activity, which could be prevented by mevalonate. This increase was comparable to the one induced by calyculin A (2 nmol/l), an inhibitor of protein phosphatase PP-1 and PP-2A. Lovastatin inhibited the expression of the PP-1 protein, which is involved in bFGF-induced DNA synthesis in cSMC. Thus, our data suggest that, lovastatin possibly affects the dephosphorylation processes of PKC and MAPK by inhibition of PP-1/PP-2A protein phosphatases which are involved in the bFGF-induced mitogenesis in cSMC.

Pex8p, an intraperoxisomal peroxin of Saccharomyces cerevisiae required for protein transport into peroxisomes binds the PTS1 receptor pex5p.

We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae. Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol. Pex8p is tightly associated with the lumenal face of the peroxisomal membrane. Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p. However, the Pex5p/Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p. The pex8Delta mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix. In pex8Delta cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event.

Protein kinase C mediates basic fibroblast growth factor-induced proliferation through mitogen-activated protein kinase in coronary smooth muscle cells.

Proliferation of coronary smooth muscle cells (cSMCs) contributes to the pathogenesis of arteriosclerosis and restenosis after angioplasty, and basic fibroblast growth factor (bFGF) is a powerful mitogen for cSMCs. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and the transcription factor c-myc in bFGF-stimulated mitogenesis, as well as the functional relationship between these factors. cSMC stimulation with bFGF resulted in phosphorylation of p42 MAPK, as well as the phosphorylation and increased expression of c-myc. The MAPK kinase (MEK) inhibitor PD98059 blocked bFGF-stimulated MAPK phosphorylation and resulted in both a decrease of c-myc expression and inhibition of bFGF-stimulated DNA synthesis in cSMCs. bFGF also increased PKC activity in cSMCs in a time-dependent manner. The inhibition of PKC by chelerythrine or its downregulation by phorbol 12-myristate 13-acetate (PMA) inhibited bFGF-induced DNA synthesis and blocked the phosphorylation of MAPK and c-myc expression in response to bFGF. This indicates an involvement of phorbol ester-sensitive PKC isoforms in MAPK activation and mitogenic signaling by bFGF. Western blot analysis revealed the presence of the phorbol ester-sensitive isoforms PKC alpha, epsilon, and gamma as well as the PKC isoforms iota, lambda, micro, and zeta in cSMCs. In this study, we show that the MAPK cascade is required for bFGF-induced proliferation and that phorbol ester-sensitive PKC isoforms contribute to the bFGF-induced cSMC mitogenesis in cSMCs.

Heparin-induced overexpression of basic fibroblast growth factor, basic fibroblast growth factor receptor, and cell-associated proteoheparan sulfate in cultured coronary smooth muscle cells.

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells (SMCs), plays a pivotal role in the pathogenesis of arteriosclerosis and restenosis. Heparin in nanogram quantities may promote or even be required for binding of bFGF to its cognate receptor. Conversely, heparin in microgram doses is a strong inhibitor of arterial SMC replication in vitro and in vivo. Bovine coronary SMCs (cSMCs) express bFGF, bFGF receptor (FGF-R1), and cell membrane-integrated proteoheparan sulfate (HSPG). These three molecules are known to form a trimolecular complex that promotes signal transduction and mitogenesis. The bFGF synthesized by cSMCs is distributed to an intracellular and a pericellular compartment. Resting cultured cells retain about 80% of their bFGF intracellularly; 20% is found in the pericellular region. During proliferation, 70% to 80% of total bFGF is expressed in the pericellular compartment. Trypsinization generates soluble forms of the complex of bFGF with the ectodomains of the bFGF receptor and cell membrane-integrated HSPG in the pericellular compartment, thus allowing quantification of pericellular bFGF by a highly specific enzyme immunoassay. Standard heparin inhibits the proliferation of cSMCs by up to 80% in a concentration range between 10 and 100 micrograms/mL medium in a dose-dependent manner but increases the protein content of cSMCs compared with proliferating control cells. The heparin-induced increase in cellular protein content includes a 60% to 100% increase in the expression of pericellular bFGF, FGF-R1, and cell membrane-integrated HSPG. Thus, under heparin treatment, the heparan sulfate side chains of cell membrane-integrated HSPG incorporate more [35S]sulfate, and the proportion of [35S]heparan sulfate among total glycosaminoglycans increases from 36% to 52%. Fluorescence-activated cell sorting analysis and [3H]thymidine incorporation experiments provide evidence for multiple effects of heparin, including blocks at early and late checkpoints of the cell cycle in heparin-treated cells. These results indicate that heparin, despite its anti-proliferative potency, stimulates the expression of all components of the bFGF system even in coronary SMCs in which growth is inhibited.

Basic fibroblast growth factor controls the expression and molecular structure of heparan sulfate in corneal endothelial cells.

Cultured bovine corneal endothelial cells express 5-8 ng basic fibroblast growth factor (bFGF)/mg cell protein and distribute it between the intracellular and pericellular compartment. Confluent cultures retain approximately 80% of the total bFGF intracellularly, whereas 20% is present in the pericellular (trypsin-releasable) compartment. No bFGF can be detected in the culture medium. The presence of 1-2 ng/ml medium of endogenous or exogenous (human recombinant) bFGF is sufficient to support cell growth. Simultaneously, cells incorporate [35S]sulfate and [3H]glucosamine into the sulfated proteoglycans associated with the cell layer at a rate that is three times higher than in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied by a shift in proteoglycan distribution. In control cells, cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans, whereas under the influence of bFGF the amount of heparan sulfate increases to approximately 60%. At the same time, the molecular structure of the heparan sulfate molecule undergoes bFGF-specific changes as indicated by the [35S]oligosaccharide pattern generated by heparitinase I degradation. The proportion of [35S]oligosaccharides with greater than six monosaccharides decreases on account of disaccharides and tetrasaccharides under the influence of bFGF. Pretreatment of bFGF with neutralizing antibodies against bFGF abolishes its biological activity. The results suggest a bFGF-dependent change in the rate of synthesis and structural features of the membrane-associated heparan sulfate in corneal endothelial cells. The modification of the heparan sulfate structure could influence its bFGF-binding and antiproliferative activity.

Growth status-dependent changes of bFGF compartmentalization and heparan sulfate structure in arterial smooth muscle cells.

Cultured bovine arterial smooth muscle cells express 6 to 7 ng basic fibroblast growth factor (bFGF)/mg cell protein and distribute it to two compartments. About 80% of total bFGF remain intracellularly, 20% are present in the pericellular (trypsin-releasable) compartment. No bFGF can be detected in the culture medium. All bFGF fractions have full biological activity. They are quantified by a highly specific immunoassay system and identified after sodium dodecyl sulfate polyacrylamide electrophoresis as a 18 kDa double band by immunoblotting. During exponential growth the intracellular concentration of bFGF decreases from about 130 pg to 20 to 40 pg/10(5) cells. Simultaneously the pericellular bFGF increases to 60-70% of total bFGF, but declines continuously with increasing cell density, whereas the intracellular bFGF reincreases under these conditions. The pericellular bFGF is known to form complexes with (membrane integrated) proteoheparan sulfate which undergoes structural changes during transition from subconfluent to confluent growth status. After metabolic labeling of the cells with [35S]sulfate and [3H]glucosamine, the 35S/3H ratio of heparan sulfate oligosaccharides increases from 1.58 during proliferation to 2.47 in growth-inhibited cells. The results indicate that the bFGF-induced proliferation of arterial smooth muscle cells depends on the pericellular localization of bFGF and on a specific molecular organization of the cell surface heparan sulfate. Depending on its specific structural characteristics heparan sulfate may promote or inhibit bFGF receptor binding and signal transduction.