SIRT3 Is a Critical Regulator of Mitochondrial Function of Fibroblasts in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

Local Complement Contributes to Pathogenic Activation of Lung Endothelial Cells in SARS-CoV-2 Infection.

Endothelial dysfunction and inflammation contribute to the vascular pathology of coronavirus disease (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells, and thus inflammation may be better explained as a secondary response to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2-infected lung alveolar epithelial cells. We found that ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) mRNA expression in lung vascular cells, including primary human lung microvascular endothelial cells (HLMVECs), pericytes, smooth muscle cells, and fibroblasts, was 20- to 90-fold lower compared with primary human alveolar epithelial type II cells. Consistently, we found that HLMVECs and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVECs when exposed to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells. Furthermore, we demonstrated that exposure of HLMVECs to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and IL-6 (interleukin 6) as well as complement components such as C3 (complement C3), C3AR1 (complement C3a receptor 1), C1QA (complement C1q A chain), and CFB (complement factor B). Taken together, our data support a model in which lung endothelial and vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells.

Immunoglobulin-driven Complement Activation Regulates Proinflammatory Remodeling in Pulmonary Hypertension.

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive.Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH.Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH.Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH.Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

Metabolic and Proliferative State of Vascular Adventitial Fibroblasts in Pulmonary Hypertension Is Regulated Through a MicroRNA-124/PTBP1 (Polypyrimidine Tract Binding Protein 1)/Pyruvate Kinase Muscle Axis.

BACKGROUND: An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments. METHODS: Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. RESULTS: We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1ß expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results. CONCLUSIONS: In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

BACKGROUND: Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). METHODS: RNA sequencing, quantitative polymerase chain reaction,13C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD+ ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. RESULTS: We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature. CONCLUSIONS: CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.

MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

RATIONALE: Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. OBJECTIVE: We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. METHODS AND RESULTS: We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti-miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract-binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3' untranslated region of polypyrimidine tract-binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. CONCLUSIONS: Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly proliferative, migratory, and inflammatory phenotype of hypertensive pulmonary adventitial fibroblasts. Thus, therapies directed at restoring miR-124 function, including histone deacetylase inhibitors, should be investigated.

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Introduction: Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. Methods: We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Results: Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Discussion: Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

The influence of synovial inflammation and hyperplasia on symptomatic outcomes up to 2 years post-operatively in patients undergoing partial meniscectomy.

OBJECTIVE: Synovitis is associated with pain and other symptoms in patients with knee osteoarthritis (OA), and in patients with meniscal tears even in the absence of radiographic OA. Patients undergoing arthroscopic partial meniscectomy were followed for 2 years to determine whether synovitis predicts post-operative symptoms. DESIGN: Thirty-three patients scheduled for arthroscopy were recruited for this pilot study. Symptoms were assessed using a knee pain scale, the Lysholm score, and the short form-12 (SF-12(®)) pre-operatively and at 16 weeks, 1 year and 2 years post-operatively. Synovial inflammation and hyperplasia were graded on surgical biopsies. Linear mixed effects models were tested to determine whether inflammation or hyperplasia is associated with outcome scores over time. RESULTS: Lysholm scores and SF-12(®) physical component sub-scores were worse pre-operatively in patients with inflammation (Lysholm: 52.42 [95% confidence interval (CI) 42.37, 62.47] vs 72.38 [66.03, 78.72], P < 0.001; SF-12: 36.81 [28.26, 45.37] vs 46.23 [40.14, 52.32], P < 0.05). Up to 2-years post-operatively, patients with inflammation achieved mean scores similar to those without inflammation. As a result, the mean improvement in Lysholm scores was 13.01 [1.48-24.53] points higher than patients without inflammation, P = 0.03. 33% (4/12) of patients with inflammation still had fair to poor Lysholm scores 2 years after surgery compared to 7% (1/15, P=0.14) without inflammation. No association between hyperplasia and symptoms was noted. CONCLUSIONS: In this pilot study of patients undergoing partial meniscectomy, synovial inflammation was associated with worse pre-operative symptoms, but not with poorer outcomes in the first 2 years post-arthroscopy. Larger cohorts and longer follow-up should be pursued to confirm this relationship, and determine if the initial response is sustained.

Emergence of fibroblasts with a proinflammatory epigenetically altered phenotype in severe hypoxic pulmonary hypertension.

Persistent accumulation of monocytes/macrophages in the pulmonary artery adventitial/perivascular areas of animals and humans with pulmonary hypertension has been documented. The cellular mechanisms contributing to chronic inflammatory responses remain unclear. We hypothesized that perivascular inflammation is perpetuated by activated adventitial fibroblasts, which, through sustained production of proinflammatory cytokines/chemokines and adhesion molecules, induce accumulation, retention, and activation of monocytes/macrophages. We further hypothesized that this proinflammatory phenotype is the result of the abnormal activity of histone-modifying enzymes, specifically, class I histone deacetylases (HDACs). Pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (termed PH-Fibs) expressed a constitutive and persistent proinflammatory phenotype defined by high expression of IL-1ß, IL-6, CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES), CCR7, CXCR4, GM-CSF, CD40, CD40L, and VCAM-1. The proinflammatory phenotype of PH-Fibs was associated with epigenetic alterations as demonstrated by increased activity of HDACs and the findings that class I HDAC inhibitors markedly decreased cytokine/chemokine mRNA expression levels in these cells. PH-Fibs induced increased adhesion of THP-1 monocytes and produced soluble factors that induced increased migration of THP-1 and murine bone marrow-derived macrophages as well as activated monocytes/macrophages to express proinflammatory cytokines and profibrogenic mediators (TIMP1 and type I collagen) at the transcriptional level. Class I HDAC inhibitors markedly reduced the ability of PH-Fibs to induce monocyte migration and proinflammatory activation. The emergence of a distinct adventitial fibroblast population with an epigenetically altered proinflammatory phenotype capable of recruiting, retaining, and activating monocytes/macrophages characterizes pulmonary hypertension-associated vascular remodeling and thus could contribute significantly to chronic inflammatory processes in the pulmonary artery wall.

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension.

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a "constitutively activated" phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the "activated" highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, α(V)ß(3) and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH.

Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension.

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

Scaling and interaction of self-similar modes in models of high Reynolds number wall turbulence.

Previous work has established the usefulness of the resolvent operator that maps the terms nonlinear in the turbulent fluctuations to the fluctuations themselves. Further work has described the self-similarity of the resolvent arising from that of the mean velocity profile. The orthogonal modes provided by the resolvent analysis describe the wall-normal coherence of the motions and inherit that self-similarity. In this contribution, we present the implications of this similarity for the nonlinear interaction between modes with different scales and wall-normal locations. By considering the nonlinear interactions between modes, it is shown that much of the turbulence scaling behaviour in the logarithmic region can be determined from a single arbitrarily chosen reference plane. Thus, the geometric scaling of the modes is impressed upon the nonlinear interaction between modes. Implications of these observations on the self-sustaining mechanisms of wall turbulence, modelling and simulation are outlined.This article is part of the themed issue 'Toward the development of high-fidelity models of wall turbulence at large Reynolds number'.

TGF-ß activation by bone marrow-derived thrombospondin-1 causes Schistosoma- and hypoxia-induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an obstructive disease of the precapillary pulmonary arteries. Schistosomiasis-associated PAH shares altered vascular TGF-ß signalling with idiopathic, heritable and autoimmune-associated etiologies; moreover, TGF-ß blockade can prevent experimental pulmonary hypertension (PH) in pre-clinical models. TGF-ß is regulated at the level of activation, but how TGF-ß is activated in this disease is unknown. Here we show TGF-ß activation by thrombospondin-1 (TSP-1) is both required and sufficient for the development of PH in Schistosoma-exposed mice. Following Schistosoma exposure, TSP-1 levels in the lung increase, via recruitment of circulating monocytes, while TSP-1 inhibition or knockout bone marrow prevents TGF-ß activation and protects against PH development. TSP-1 blockade also prevents the PH in a second model, chronic hypoxia. Lastly, the plasma concentration of TSP-1 is significantly increased in subjects with scleroderma following PAH development. Targeting TSP-1-dependent activation of TGF-ß could thus be a therapeutic approach in TGF-ß-dependent vascular diseases.

Risk factors for Amphotericin B-associated nephrotoxicity.

PURPOSE: A case-control study was performed to identify and quantify risk factors for amphotericin B-associated nephrotoxicity. PATIENTS AND METHODS: Thirty-five patients receiving intravenous amphotericin B for treatment of proven or suspected fungal infection who developed nephrotoxicity (greater than 100% increase in baseline serum creatinine to a level above the normal range) were compared with 60 control patients receiving amphotericin B who did not develop nephrotoxicity. Amphotericin B dosing variables and other potential risk factors were analyzed in a logistic regression model. RESULTS: Cases of nephrotoxicity received a significantly higher average daily dose of amphotericin B (0.49 +/- 0.18 mg/kg/day) than did controls (0.34 +/- 0.17 mg/kg/day). In a multivariate model, the risk of nephrotoxicity increased 3.7-fold for each 50-mg increase in total dose for a fixed duration of therapy and patient weight. Risk decreased by a factor of 0.4 for each extra day of therapy for a fixed total dose and weight. An increase in weight was also protective when the two other dosage variables were held constant. Each 0.10 mg/kg/day dose increment was associated with a 1.8-fold (95% confidence interval, 1.2 to 2.7) increase in the risk of nephrotoxicity. Other significant risk factors included diuretic use during amphotericin B therapy (12.5, 1.7 to 94.7), for which a linear dose-response relationship was demonstrated, and an abnormal baseline serum creatinine level (15.4, 1.4 to 173.2). CONCLUSION: Risk factors for amphotericin B-associated nephrotoxicity include higher average daily doses (approximately a doubling for each 0.10 mg/kg/day increment), diuretic use, and abnormal baseline renal function. These data suggest possible protective interventions and will aid clinicians in assessing the risk-benefit ratio of amphotericin B therapy for deep fungal infection.

Magnetic resonance imaging of relative glycosaminoglycan distribution in patients with autologous chondrocyte transplants.

RATIONALE AND OBJECTIVES: Autologous chondrocyte transplantation (ACT) is a potential treatment for full-thickness chondral lesions in the knee. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) has recently been developed as a sensitive and specific measure of cartilage glycosaminoglycans (GAGs). Under the conditions of dGEMRIC, T1 is directly related to the GAG concentration. Our aim for this study was to demonstrate the potential of dGEMRIC to evaluate ACT implants. METHODS: Eleven ACT implants were studied 2 to 24 months postoperatively by dGEMRIC. T1 values from three regions of interest were obtained to examine GAG content (1) in the implant, (2) in native cartilage adjacent to the implant, and (3) in native cartilage further removed from the implant (as "control"). RESULTS: One implant failed and therefore was not included. Four of the implants were studied between 2 and 6 months postoperatively and showed low T1 (GAG), less than 80% of the control native cartilage. Five of the six implants studied between 12 and 24 months postoperativley showed T1 (GAG) comparable to (>80%) of control. One 18-month graft showed low T1 comparable to the surrounding native cartilage, with normal GAG seen in cartilage far from the graft site. The GAG index (T1 values of the graft normalized to control) from the group of implants 6 months or less was 59% +/- 5% of control, whereas those at 12 to 24 months were 91% +/- 18% of control. The two groups were statistically different with a P value of 0.005. CONCLUSIONS: The GAG level in grafts that were implanted for less than 12 months appeared to be lower than that in the remote cartilage. At 12 months or greater, the grafts in this study had GAG levels that were comparable to both the adjacent and remote cartilage. This preliminary study of ACT implants has shown that it is feasible to apply the dGEMRIC technique in patients with ACT as a way to obtain information related to the composition of grafts. These results provide motivation and the pilot data with which to design further clinical studies.

Coupling between human muscle spindle endings and motor units assessed using spike-triggered averaging.

The electromyographic activity (EMG) generated by voluntary contraction of a muscle was averaged using the potentials from 18 identified muscle spindle afferents as a trigger. In post-spike averages of 1000-10,000 sweeps, no evidence of reflex excitation of the homonymous motoneurone pool was detected. In pre-spike averages there was no evidence of a motor-unit EMG potential that was closely correlated to the trigger spike. A single spindle afferent has only a weak reflex effect on an active motoneurone pool and must be part of a synchronized volley to affect motoneurone discharge significantly. No evidence was found for spindle activation via beta-motoneurones in weak voluntary contractions.

Mechanical evaluation of a soft tissue interference screw in free tendon anterior cruciate ligament graft fixation.

In this study of bioabsorbable screw fixation of free tendon grafts used in anterior cruciate ligament reconstruction, we performed load-to-failure and cyclic loading of tendon fixation in porcine bone. Bone density measurements from dual photon absorptometry scans were obtained to correlate bone density with fixation failure. The average density of porcine bone (1.42 g/cm2) was similar to that of young human bone (1.30 g/cm2) and significantly higher than that of elderly human cadaveric bone specimens (0.30 g/cm2). Cyclic loading was performed on free tendon grafts fixed with a bioabsorbable screw alone and on grafts fixed with a bioabsorbable screw and an anchor (polylactic acid ball or cortical bone disk). Stiffness of fixation increased substantially with the addition of a cortical bone disk anchor or polylactic acid ball compared with the interference screw alone. Tensile fixation strength of central quadriceps free tendon and hamstring tendon grafts were significantly superior in porcine bone of density similar to young human bone than in elderly human cadaveric bone. The bioabsorbable interference screw yielded loads at failure comparable with traditional bone-tendon-bone and hamstring tendon fixation when controlled for bone density. The addition of a cortical bone disk anchor provided the most optimal fixation of free tendon with the bioabsorbable screw and reduced slippage with cyclic loading to a very low level.

The near-neutral atmospheric surface layer: turbulence and non-stationarity.

The neutrally stable atmospheric surface layer is used as a physical model of a very high Reynolds number, canonical turbulent boundary layer. Challenges and limitations with this model are addressed in detail, including the inherent thermal stratification, surface roughness and non-stationarity of the atmosphere. Concurrent hot-wire and sonic anemometry data acquired in Utah's western desert provide insight to Reynolds number trends in the axial velocity statistics and spectra.

Asymptotic scaling in turbulent pipe flow.

The streamwise velocity component in turbulent pipe flow is assessed to determine whether it exhibits asymptotic behaviour that is indicative of high Reynolds numbers. The asymptotic behaviour of both the mean velocity (in the form of the log law) and that of the second moment of the streamwise component of velocity in the outer and overlap regions is consistent with the development of spectral regions which indicate inertial scaling. It is shown that an 'inertial sublayer' in physical space may be considered as a spatial analogue of the inertial subrange in the velocity spectrum and such behaviour only appears for Reynolds numbers R+>5 x 10(3), approximately, much higher than was generally thought.

Component of muscle spindle discharge related to arterial pulse.

1. In human subjects microelectrode recordings were made from 25 muscle spindle afferents from the pretibial muscles. 2. The spike discharges of three endings were locked in time to the arterial pulse. With 17 of the remaining endings, there was a significant pulse-related modulation of discharge rate. For these 20 endings the latency to the onset of the pulse-related influence was 200-310 ms. 3. The time course of the modulation of discharge rate was similar to that of arterial blood flow, as estimated using a Doppler flowmeter. With four endings occlusion of blood flow using a sphygmomanometer cuff reduced any modulation. 4. For five endings the contribution by the arterial modulation to the variance of discharge of the ending was 3-54%. For the population of endings there was no significant relationship between the depth of modulation and coefficient of variation. 5. It is concluded that the arterial pulse can be significant contributor to the variability of muscle spindle discharge. The pulsatile effects seen in the responses of single afferents are unlikely to be eliminated in the summed activity forming the population response. This could constitute a limitation of the information capacity of the population of muscle spindle afferents.