Multicomponent telerehabilitation programme for older veterans with multimorbidity: a programme evaluation.

INTRODUCTION: Older veterans with multimorbidity experience physical, mental and social factors which may negatively impact health and healthcare access. Physical function, behaviour change skills and loneliness may not be addressed during traditional physical rehabilitation. Thus, a multicomponent telerehabilitation programme could address these unmet needs. This programme evaluation assessed the safety, feasibility and change in patient outcomes for a multicomponent telerehabilitation programme. METHODS: Individuals were eligible if they were a veteran/spouse, age ≥50 years and had ≥3 comorbidities. The telerehabilitation programme included four core components: (1) High-intensity rehabilitation, (2) Coaching interventions, (3) Social support and (4) Technology. Physical therapists delivered the 12-week programme and collected patient outcomes at baseline, 4 weeks, 8 weeks and 12 weeks. Programme evaluation measures included safety events (occurrence and type), feasibility (adherence) and patient outcomes (physical function). Safety and feasibility outcomes were analysed using descriptive statistics. The mean pre-post programme difference and 95% CI for patient outcomes were generated using paired t-tests. RESULTS: Twenty-one participants enrolled in the telerehabilitation programme; most were male (81%), white (72%) and non-Hispanic (76%), with an average of 5.7 (3.0) comorbidities. Prevalence of insession safety events was 3.2% (0.03 events/session). Fifteen (71.4%) participants adhered to the programme (attended ≥80% of sessions). Mean (95% CI) improvements for physical function are as follows: 4.7 (2.4 to 7.0) repetitions for 30 s sit to stand, 6.0 (4.0 to 9.0) and 5.0 (2.0 to 9.0) repetitions for right arm curl and left arm curl, respectively, and 31.8 (15.9 to 47.7) repetitions for the 2 min step test. CONCLUSION: The telerehabilitation programme was safe, feasible and demonstrated preprogramme to postprogramme improvements in physical function measures while addressing unmet needs in a vulnerable population. These results support a randomised clinical trial while informing programme and process adaptations.

Clinician and staff perspectives on potential disparities introduced by the rapid implementation of telehealth services during COVID-19: a mixed-methods analysis.

The COVID-19 pandemic has rapidly altered ambulatory health care delivery and may have worsened disparities in health care access. To assess the telehealth implementation experiences of ambulatory personnel in different disciplines and their perspectives on potential telehealth disparities, and to make recommendations for more equitable telehealth delivery. We used a convergent parallel mixed-methods design. Clinic managers from geriatric medicine, internal medicine, and psychiatry e-mailed a survey to clinicians and staff regarding experiences with telehealth care delivery. Quantitative survey responses were analyzed with Fisher's Exact tests. Qualitative responses were coded thematically. Recommendations were categorized by type of implementation strategy. Quantitative and qualitative findings on telehealth disparities were merged in a joint data display. Respondents (n = 147, 57% response rate) were distributed across three specialties: 66% internal medicine, 19% psychiatry, and 14% geriatric medicine. Prior to 2020, 77% of clinicians had never delivered telehealth services. By Spring 2020, 78% reported conducting more than half of clinic visits by telehealth. Among clinicians, 52% agreed/strongly agreed that rapid telehealth implementation exacerbated access to care disparities to: older adult patients, those with limited internet access, and those needing interpretation services. Staff expressed similar difficulties with telehealth set-up especially for these patients. To improve telehealth equity, clinicians recommended to: (i) change infrastructure; (ii) train and educate stakeholders; and (iii) support clinicians. Clinicians and staff reported specific subpopulations had challenges in accessing telehealth visits. To avoid perpetuating telehealth access disparities, further co-discovery of equitable implementation strategies with patients and clinics are urgently needed.

Reactive oxygen species mediate high glucose-induced heparanase-1 production and heparan sulphate proteoglycan degradation in human and rat endothelial cells: a potential role in the pathogenesis of atherosclerosis.

AIMS/HYPOTHESIS: The content of heparan sulphate is reduced in the endothelium under hyperglycaemic conditions and may contribute to the pathogenesis of atherosclerosis. Heparanase-1 (HPR1) specifically degrades heparan sulphate proteoglycans. We therefore sought to determine whether: (1) heparan sulphate reduction in endothelial cells is due to increased HPR1 production through increased reactive oxygen species (ROS) production; and (2) HPR1 production is increased in vivo in endothelial cells under hyperglycaemic and/or atherosclerotic conditions. METHODS: HPR1 mRNA and protein levels in endothelial cells were analysed by RT-PCR and Western blot or HPR1 enzymatic activity assay, respectively. Cell surface heparan sulphate levels were analysed by FACS. HPR1 in the artery from control rats and a rat model of diabetes, and from patients under hyperglycaemic and/or atherosclerotic conditions was immunohistochemically examined. RESULTS: High-glucose-induced HPR1 production and heparan sulphate degradation in three human endothelial cell lines, both of which were blocked by ROS scavengers, glutathione and N-acetylcysteine. Exogenous H(2)O(2) induced HPR1 production, subsequently leading to decreased cell surface heparan sulphate levels. HPR1 content was significantly increased in endothelial cells in the arterial walls of a rat model of diabetes. Clinical studies revealed that HPR1 production was increased in endothelial cells under hyperglycaemic conditions, and in endothelial cells and macrophages in atherosclerotic lesions. CONCLUSIONS/INTERPRETATION: Hyperglycaemia induces HPR1 production and heparan sulphate degradation in endothelial cells through ROS. HPR1 production is increased in endothelial cells from a rat model of diabetes, and in macrophages in the atherosclerotic lesions of diabetic and non-diabetic patients. Increased HPR1 production may contribute to the pathogenesis and progression of atherosclerosis.

Expansion of HIV-1 Screening and Anti-Retroviral Treatment Programs in a Resource-Poor Setting: Results from a Faith-Based Organization in Jos; Plateau State; Nigeria

Background: Until recently; availability of anti-retroviral therapy (ART) in Nigeria has been limited to government and university based programs. Through the United States' President's Emergency Plan For AIDS Relief (PEPFAR); additional funding has become available for the treatment of HIV-positive patients. Objective: To report the expansion of HIV-1 screening; enrollment in an ART program; and treatment outcomes over twelve months among HIV-positive patients at a nonprofit; non-governmental faith-based clinic providing free and holistic care in Jos City; Plateau State; Nigeria. Design: This was a retrospective analysis of HIV-1 screening and ART received by patients at Faith Alive Foundation Hospital (FAFH). From January through December 2005; voluntary counseling and testing (VCT) was freely available to all patients who requested it. Also beginning in December 2004; HIV-1 infected patients were enrolled in an expanded HIV/AIDS program at FAFH; where patients in clinical stages 3 or 4 based on World Health Organiza- tion (WHO) clinical classification; or had a CD4+ cell count of less than 200/ ml; were initiated on a simplified highly active ART (HAART) regimen through PEPFAR funding. Data from the first six quarters of the FAFH-PEPFAR ART program are presented here. Additionally; analysis of 645 patients who have received ART for one year; including changes in CD4+ cell count from baseline is shown. Results: In 2005; a total of 7672 persons received VCT and 3869 (50.4) HIV-1 positive results were found. From October 2004 to March 2006; the total number of patients enrolled in the FAFH HIV/AIDS program rose from 1330 to 5946 people. Over the same period; the number of patients who received ART increased from 302 to 1667. A majority of patients received an oral ART regimen consisting of generic nevirapine; lamivudine; and stavudine. The number of patients initiated on ART each quarter ranged from 57-578; and the number of deaths of patients on ART was between 12 and 21 people per quarter. Analysis of 645 patients initiated on ART during the first quarter of the FAFH-PEPFAR ART program revealed that the median CD4+ cell count at baseline was 106/ml and the median increase was 173/ml (median time of follow-up; 7.0 months). Conclusions: Comprehensive HIV/AIDS care has been significantly expanded at FAFH; a free and holistic medical center in Jos; Nigeria. Cumulative data from the first year of this PEPFAR-funded ART program indicated that promising outcomes are achievable through delivery of care at this faith-based medical center

6-Hydroxydopamine-induced alterations in blood-brain barrier permeability.

Vascular inflammation is well known for its ability to compromise the function of the blood--brain barrier (BBB). Whether inflammation on the parenchymal side of the barrier, such as that associated with Parkinson's-like dopamine (DA) neuron lesions, similarly disrupts BBB function, is unknown. We assessed BBB integrity by examining the leakage of FITC-labeled albumin or horseradish peroxidase from the vasculature into parenchyma in animals exposed to the DA neurotoxin 6-hydroxydopamine (6OHDA). Unilateral injections of 6OHDA into the striatum or the medial forebrain bundle produced increased leakage in the ipsilateral substantia nigra and striatum 10 and 34 days following 6OHDA. Microglia were markedly activated and DA neurons were reduced by the lesions. The areas of BBB leakage were associated with increased expression of P-glycoprotein and beta 3-integrin expression suggesting, respectively, a compensatory response to inflammation and possible angiogenesis. Behavioural studies revealed that domperidone, a DA antagonist that normally does not cross the BBB, attenuated apomorphine-induced stereotypic behaviour in animals with 6OHDA lesions. This suggests that drugs which normally have no effect in brain can enter following Parkinson-like lesions. These data suggest that the events associated with DA neuron loss compromise BBB function.

Hydrogen peroxide induces a rapid production of nitric oxide in mung bean (Phaseolus aureus).

Nitric oxide (NO) has recently been identified as an important signaling molecule in plant immune response. The present study aims to investigate the signaling pathway that leads to NO production. Using the NO specific fluorescent dye DAF-2DA, we observed rapid production of NO in mung bean leaves after the addition of 10 mM hydrogen peroxide (H(2)O(2)). NO was probably produced by a NOS-like enzyme in plants, as the NO production was inhibited by l-NAME, a NOS inhibitor. The NOS-like activity in the total leaf protein preparation of mung bean (Phaseolus aureus) was elevated 8.3-fold after 10 mM H(2)O(2) treatment, as demonstrated using the chemiluminescence NOS assay. The NOS-like activity was BH(4) dependent: omitting BH(4) in the reaction mixture of NOS assay reduced the NOS activity by 76%. We also found that the H(2)O(2) induced NO production was mediated via calcium ion flux, as it was blocked in the presence of a calcium ion channel blocker, verapamil. Results from the present study identified H(2)O(2) as an upstream signal that leads to NO production in plants. H(2)O(2) and NO, besides acting as two independent signaling molecules in plant immune response, may interrelate to form an oxidative cell death (OCD) cycle.

Update on pulmonary edema: the role and regulation of endothelial barrier function.

Discovery of the pathophysiologic mechanisms leading to pulmonary edema and identification of effective strategies for prevention remain significant clinical concerns. Endothelial barrier function is a key component for maintenance of the integrity of the vascular boundary in the lung, particularly since the gas exchange surface area of the alveolar-capillary membrane is large. This review is focused on new insights in the pulmonary endothelial response to injury and recovery, reversible activation by edemagenic agents, and the biochemical/structural basis for regulation of endothelial barrier function. This information is discussed in the context of fundamental concepts of lung fluid balance and pulmonary function.

Protein phosphatase 2B inhibitor potentiates endothelial PKC activity and barrier dysfunction.

Serine/threonine (Ser/Thr) protein phosphatases (PPs) are implicated in the recovery from endothelial barrier dysfunction caused by inflammatory mediators. We hypothesized that Ser/Thr PPs may regulate protein kinase C (PKC), a critical signaling molecule in barrier dysfunction, in the promotion of barrier recovery. Western analysis indicated that bovine pulmonary microvascular endothelial cells (BPMECs) expressed the three major Ser/Thr PPs, PP1, PP2A, and PP2B. Pretreatment with 100 ng/ml of FK506 (a PP2B inhibitor) but not with the PP1 and PP2A inhibitors calyculin A or okadaic acid potentiated the thrombin-induced increase in PKC phosphotransferase activity. FK506 also potentiated thrombin-induced PKC-alpha but not PKC-beta phosphorylation. FK506 but not calyculin A or okadaic acid inhibited recovery from the thrombin-induced decrease in transendothelial resistance. Neither FK506 nor okadaic acid altered the thrombin-induced resistance decrease, whereas calyculin A potentiated the decrease. Downregulation of PKC with phorbol 12-myristate 13-acetate rescued the FK506-mediated inhibition of recovery, which was consistent with the finding that the thrombin-induced phosphorylation of PKC-alpha was reduced during the recovery phase. These results indicated that PP2B may play a physiologically important role in returning endothelial barrier dysfunction to normal through the regulation of PKC.

The human HMGB1 promoter is modulated by a silencer and an enhancer-containing intron.

The highly conserved, ubiquitous high mobility group protein HMGB1 (formerly named as HMG1) is an architectural transcription factor encoded by a single functional gene in human. HMGB1 is expressed in almost all cell or tissue types studied. In general, it is expressed at a basal level in most cells but at a slightly elevated level of 2--3-fold in actively proliferating tissues or estrogen stimulated breast cancer cells. To understand the regulatory mechanism controlling expression of the human HMGB1 gene, we cloned and analyzed the upstream region as well as the first intron of this gene. We found that transcription of the human HMGB1 gene in the breast cancer MCF-7 cells starts at one major site 57 nucleotides upstream from the first exon-intron boundary. Expression of the human HMGB1 gene is under the control of a very strong TATA-less promoter, which has an activity more than 18-fold that of the SV40 promoter. Immediately upstream, a silencer element is present. This silencer can repress the activity of the HMGB1 promoter down to just one-sixth. The first intron of the human HMGB1 gene contains enhancer elements, which can increase the human HMGB1 promoter activity by 2--3-fold. We postulate that the human HMGB1 gene is capable of being expressed at a very high level. The basal level of expression observed in most cells is probably a result of the strong promoter being held in check by the silencer. The 2--3-fold increase in HMGB1 expression observed in proliferating cells or breast cancer cells stimulated by estrogen may probably result from the action of the enhancer elements in intron 1.

Oxidant stress and endothelial cell dysfunction.

Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels, ROS can function as signaling molecules participating as signaling intermediates in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidant stress, playing a critical role in the pathophysiology of several vascular diseases and disorders. Specifically, oxidant stress increases vascular endothelial permeability and promotes leukocyte adhesion, which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-kappaB. This review discusses recent findings on the cellular and molecular mechanisms by which ROS signal events leading to impairment of endothelial barrier function and promotion of leukocyte adhesion. Particular emphasis is placed on the regulation of cell-cell and cell-surface adhesion molecules, the actin cytoskeleton, key protein kinases, and signal transduction events.

The chicken genome contains no HMG1 retropseudogenes but a functional HMG1 gene with long introns.

We have cloned the genomic sequence coding for the high mobility group 1 (HMG1) protein in chickens. Multiple sequence alignment shows that the chicken HMG1 gene is highly homologous to the human and the mouse HMG1 genes. The gene structure of chicken HMG1 is similar to that of the mouse and the human HMG1 genes, with the same exon-intron boundaries. However, in contrast to other avian genes that have shorter introns, the chicken HMG1 gene has introns that are twice as long as their mammalian homologues. In addition to the functional, intron-containing HMG1 gene, all mammalian genomes contain more than 50 copies of HMG1 retropseudogenes each, while in the chicken genome there are no HMG1 retropseudogenes. This finding suggests that the HMG1 retropseudogenes arose in mammals after their divergence away from the birds.

Regulation of endothelial barrier function by the cAMP-dependent protein kinase.

Elevation of cAMP promotes the endothelial cell (EC) barrier and protects the lung from edema development. Thus, we tested the hypothesis that both increases and decreases in PKA modulate EC function and coordinate distribution of regulatory, adherence, and cytoskeletal proteins. Inhibition of PKA activity by RpcAMPS and activation by cholera toxin was verified by assay of kemptide phosphorylation in digitonin permeabilized EC. Inhibition of PKA by RpcAMPS or overexpression of the endogenous inhibitor, PKI, decreased monolayer electrical impedance and exacerbated the decreases produced by agonists (thrombin and PMA). RpcAMPS directly increased F-actin content and organization into stress fibers, increased co-staining of actin with both phosphatase 2B and myosin light chain kinase (MLCK), caused reorganization of focal adhesions, and decreased catenin at cell borders. These findings are similar to those evoked by thrombin. In contrast, cholera toxin prevented the agonist-induced resistance decrease and protein redistribution. Although PKA activation attenuated thrombin-induced myosin light chain (MLC) phosphorylation, PKA inhibition per se did not cause MLC phosphorylation or affect [Ca2+]i. These studies indicate that a decrease in PKA activity alone can produce disruption of barrier function via mechanisms not involving MLCK and support a central role for cAMP/PKA in regulation of cytoskeletal and adhesive protein function in EC which correlates with altered barrier function.

Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.

We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.

Protein kinase C beta modulates thrombin-induced Ca2+ signaling and endothelial permeability increase.

We investigated the function of the Ca2+-dependent protein kinase C (PKC) beta1 in the regulation of endothelial barrier property. Human dermal microvascular endothelial cells (HMEC-1) were transduced with full-length PKCbeta1 antisense (AS) cDNA or control pLNCX vector to generate stable cell lines (HMEC-AS and HMEC-pLNCX, respectively). Analyses indicated that HMEC-AS expressed the antisense PKCbeta1 transcript with decreased PKCbeta protein level (without a change in PKCalpha or PKCepsilon). The baseline transendothelial 125I-albumin clearance rates of HMEC-1, HMEC-pLNCX, and HMEC-AS were 5.0+/-0.5 x 10(-2), 6.8+/-0.4 x 10(-2), and 6.9+/-0.6 x 10(-2) microl/min, respectively. Activation of HMEC-1 and HMEC-pLNCX with phorbol 12-myristate 13-acetate (PMA) increased the rates to the respective 14.5+/-1.7 x 10(-2) microl/min and 16.9+/-2.8 x 10(-2) microl/min (corresponding to 191% and 149% increases over baseline). However, in HMEC-AS, PMA increased the rate to 9.8+/-1.0 x 10(-2) microl/min (42%). When HMEC-1 and HMEC-pLNCX were activated with thrombin, the rates increased to 10.8+/-1.4 x 10(-2) and 14.0+/-1.9 x 10(-2) microl/min, respectively (116% and 106%). In contrast, thrombin stimulation of HMEC-AS more than doubled the increase to 27.2+/-3.5 x 10(-2) microl/min (294%). Furthermore, the thrombin-induced peak increase in the [Ca2+]i in HMEC-AS was greater than in control cells. Fluorescence-activated cell sorter analysis of thrombin receptor expression indicated that the augmented thrombin-induced responses were not attributable to altered receptor density in HMEC-AS. These results indicate that PKCbeta functions in a negative feedback manner to inactivate thrombin-generated signals and thereby modulates the endothelial permeability increase. Because decreased PKCbeta expression significantly reduced the PMA-induced permeability increase, PKCbeta may downregulate thrombin receptor function upstream of PKC activation (i.e., Ca2+).

Protein kinase C beta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells.

We studied the effects of protein kinase C (PKC) activation on endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration in human dermal microvascular endothelial cells (HMEC) exposed to alpha-thrombin or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TPA reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicating heterologous PAR-1 desensitization. Treatment of cells with staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC beta isozymes (PKC beta I and PKC beta II) by transducing cells with antisense cDNA of PKC beta I prevented the TPA-induced decrease in cell surface PAR-1 expression and restored approximately 60% of the cytosolic Ca2+ signal in response to thrombin. In contrast, depletion of PKC beta isozymes did not affect the loss of cell surface PAR-1 and induction of homologous PAR-1 desensitization by thrombin. Therefore, homologous PAR-1 desensitization by thrombin occurs independently of PKC beta isozymes, whereas the PKC beta-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

Leishmania lipophosphoglycan reduces monocyte transendothelial migration: modulation of cell adhesion molecules, intercellular junctional proteins, and chemoattractants.

We previously identified the structural requirement for the inhibitory activity of Leishmania lipophosphoglycan (LPG) to block endothelial adhesion to monocytes. Here we showed that LPG reduces transendothelial migration of monocytes. LPG pretreatment of endothelial cells (2 microM, 1 h) reduced monocyte migration across endothelial cells activated by bacterial endotoxin (LPS) or IL-1beta (60 and 46%, respectively). A fragment of LPG (i.e., repeating phosphodisaccharide (consisting of galactosyl-mannose)) and LPG coincubated with LPG-neutralizing mAb lacks inhibitory activity on monocyte migration. Pretreatment of monocytes with LPG (2 microM, 1 h) also did not affect monocyte migration through control or LPS-activated endothelial cells. FACS analysis reveals that LPG treatment blocked the LPS-mediated expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells and monocyte adhesion without altering the integrity of the endothelial monolayer. LPG (2 microM, 1 h) alone was capable of altering the expression and distribution of two junctional adhesion molecules, CD31 and vascular endothelium cadherin, as well as reversing the effects of LPS on these proteins. The induction of endothelial cells by LPS to transcribe and release monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by LPG (40-65%). LPG treatment of nonactivated endothelial cells also suppressed by 55 to 75% the monocyte migration triggered by a MCP-1 chemoattractant gradient, and coincubation of LPG with neutralizing mAb abrogated the inhibitory activity. Together, these data point to a novel anti-inflammatory function of LPG in reducing monocyte migration across endothelial cells via a mechanism of inhibition of endothelial expression of cell adhesion molecules, modulation of intercellular junctional proteins, and synthesis of MCP-1.

Site-specific thrombin receptor antibodies inhibit Ca2+ signaling and increased endothelial permeability.

Thrombin receptor is activated by thrombin-mediated cleavage of the receptor's NH2 terminus between Arg-41 and Ser-42, generating a new NH2 terminus that functions as a "tethered ligand" by binding to sites on the receptor. We prepared antibodies (Abs) directed against specific receptor domains to study the tethered ligand-receptor interactions required for signaling the increase in endothelial permeability to albumin. We used polyclonal Abs directed against the peptide sequences corresponding to the extracellular NH2 terminus [residues 70-99 (AbDD) and 1-160 (AbEE)] and extracellular loops 1 and 2 [residues 161-178 (AbL1) and 244-265 (AbL2)] of the seven-transmembrane thrombin receptor. Receptor activation was determined by measuring changes in cytosolic Ca2+ concentration ([Ca2+]i) in human dermal microvascular endothelial cells (HMEC) loaded with Ca(2+)-sensitive fura 2-acetoxymethyl ester dye. The transendothelial 125I-labeled albumin clearance rate (a measure of endothelial permeability) was determined across the confluent HMEC monolayers. AbEE (300 micrograms/ml), directed against the entire extracellular NH2-terminal extension, inhibited the thrombin-induced increases in [Ca2+]i and the endothelial 125I-albumin clearance rate (> 90% reduction in both responses). AbDD (300 micrograms/ml), directed against a sequence within the NH2-terminal extension, inhibited 70% of the thrombin-induced increase in [Ca2+]i and 60% of the increased 125I-albumin clearance rate. AbL2 (300 micrograms/ml) inhibited these responses by 70 and 80%, respectively. However, AbL1 (300 micrograms/ml) had no effect on either response. We conclude that NH2-terminal extension and loop 2 are critical sites for thrombin receptor activation in endothelial cells and thus lead to increased [Ca2+]i and transendothelial permeability to albumin.

Bradykinin- and thrombin-induced increases in endothelial permeability occur independently of phospholipase C but require protein kinase C activation.

We determined whether activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and a subsequent increase in cytosolic calcium concentration ([Ca2+]i) was an obligatory signaling event mediating the increase in transendothelial permeability induced by bradykinin (BK) and alpha-thrombin (alpha-T). Both BK and alpha-T (each at a concentration range of 0.01-1 microM) caused dose-dependent increases in transendothelial 125I-albumin permeability in cultured bovine pulmonary artery endothelial cell monolayers. Both agonists also produced a rise in inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] by 10 sec that was followed by a prolonged increase in [Ca2+]i. Pretreatment of endothelial cells with the PLC inhibitor, 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dion [(U73122) at 10 microM for 15 min], prevented the increases in Ins(1,4,5)P3 and [Ca2+]i induced by both BK and alpha-T. However, inhibition of PLC with U73122 or another PLC inhibitor, neomycin, did not prevent the increase in endothelial permeability induced by either agonist. In contrast, depletion of cellular protein kinase C (PKC) with phorbol-12-myristate 13-acetate (0.01 microM for 20 hr) increased both BK- and alpha-T-induced phosphoinositide turnover but inhibited the agonist-induced increase in permeability. A PKC inhibitor, staurosporine (5 microM) likewise inhibited the BK-induced increase in endothelial cell permeability to albumin. We conclude that increases in endothelial permeability induced by the inflammatory mediators, BK and thrombin, can occur independently of PLC activation and increased [Ca2+]i but that a PKC-dependent pathway is required for the permeability response.

Mechanisms of increased endothelial permeability.

The increase in endothelial permeability in response to inflammatory mediators such as thrombin and histamine is accompanied by reversible cell rounding and interendothelial gap formation, suggesting that the predominant transport pathway is a diffusive one (i.e., via cellular junctions (paracellular transport)). However, vesicle-mediated transport (i.e., via albumin-binding protein gp60) may also contribute significantly to the overall increase in permeability. Regulation of paracellular transport in endothelial cells is associated with modulation of actin-based systems, which anchor the cell to its neighbor or extracellular matrix, thus maintaining endothelial integrity. At the cell-cell junctions, actin is linked indirectly to the plasma membrane by linking proteins (e.g., vinculin, catenins, alpha-actinin) to cadherins, which function in homophilic intercellular adhesion. At endothelial focal contacts, the transmembrane receptors (integrins) for matrix proteins are linked to actin via linking proteins (i.e., vinculin, talin, alpha-actinin). In response to inflammatory mediators, second messengers signal two regulatory pathways, which modulate the actin-based systems, and can thus lead to impairment of the endothelial barrier integrity. One critical signal may be based on protein kinase C isoenzyme specific phosphorylation of linking proteins at the cell-cell and cell-matrix junctions. The increased phosphorylation is associated with actin reorganization, cell rounding, and increased paracellular transport. Another important event is the activation of myosin light chain kinase (MLCK), which causes an actin-myosin-based contraction that may lead to centripetal retraction of endothelial cells. Current research is being conducted at identification of protein substrates of protein kinase C isoenzymes, the specific role of their phosphorylation in barrier function, and determination of the precise role of MLCK in modulation of endothelial barrier function. Since mechanisms by which the increased permeability is returned to normal may be regulated at multiple levels (e.g., receptor desensitization, protein kinase C mediated negative feedback pathways, activation of protein phosphatases), it is also important to determine these cellular "off-switch" mechanisms.