Changes and Associations Between Gait Biomechanics and Knee Inflammation After Aspiration and Glucocorticoid Injection for Knee Osteoarthritis.

OBJECTIVE: Although knee inflammation is thought to adversely affect joint function in patients with knee osteoarthritis (OA), the effects of reducing knee inflammation on gait biomechanics and strength are unknown. Our objectives were to compare ultrasound (US) measures of knee inflammation, gait biomechanics, knee extension and flexion strength, and pain before and after knee aspiration and glucocorticoid injection, and to explore associations among changes. METHODS: Forty-nine patients (69 knees) with symptomatic knee OA and synovitis were tested before and 3-4 weeks after US-guided knee aspiration and glucocorticoid injection. At each visit, participants completed US assessments for inflammatory features of knee OA, 3D gait analysis, isokinetic knee extension and flexion strength tests, and Knee Osteoarthritis Outcome Score (KOOS) pain subscales. Linear and polynomial mixed-effects regression models were used to investigate changes and their associations. RESULTS: Changes were observed for the synovitis score (unstandardized ß [post-injection minus pre-injection] -0.55/9 [95% confidence interval (95% CI) -0.97, -0.12]), effusion depth (-1.05 mm [95% CI -1.07, -0.39]), KOOS pain (unstandardized ß 5.91/100 [95% CI 1.86, 9.97]), peak external knee flexion and extension moments (KFM; 3.33 Nm [95% CI 0.45, 6.22]), KEM (-2.99 Nm [95% CI -5.93, -0.05]), and knee extension strength (4.70 Nm [95% CI 0.39, 9.00]) and flexion strength (3.91 Nm [95% CI 1.50, 6.81]). The external KFM increased during 13-38% and 76-89% of stance post-injection. When controlled for time, greater synovitis was associated with lower knee extension strength, while lower pain was associated with increased knee extension and flexion strength. CONCLUSION: In patients with knee OA and synovitis, reduced inflammation and pain after aspiration and glucocorticoid injection are associated with changes in knee gait biomechanics and strength.

Synovitis Is Associated With Constant Pain in Knee Osteoarthritis: A Cross-sectional Study of OMERACT Knee Ultrasound Scores.

OBJECTIVE: To examine the association between ultrasound (US)-detected knee inflammation and intermittent and constant pain experiences in patients with knee osteoarthritis (OA). METHODS: Participants with radiographically early-stage (Kellgren-Lawrence arthritis grading scale [KL] ≤ 2) and late-stage (KL ≥ 3) disease and frequent symptoms underwent musculoskeletal US measures of inflammation using the Outcome Measures in Rheumatology (OMERACT) knee US scoring system. Pain experiences were captured using the Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP) tool. We assessed the association between US-synovitis and ICOAP pain experiences using a series of linear, logistic, or multinomial logistic regression models (as appropriate for each variable), while adjusting for age, sex, BMI, and radiographic stage. Secondary analyses were performed similarly by radiographic stage. RESULTS: Pain and synovitis measures from 248 patients (453 knees) were included. Worse synovitis was associated with higher ICOAP constant pain scores (ß 8.05, 95% CI 0.67-15.43), but not intermittent pain scores. Moderate-to-severe synovitis was associated with a 4.73-fold increased relative risk (95% CI 1.06-17.00) of a constant pain pattern. In secondary analyses, moderate-to-severe synovitis in early radiographic OA was associated with 2.70-higher odds (95% CI 1.04-7.02) of any constant pain, 3.28-higher odds (95% CI 1.43-7.52) of any intermittent pain, and with higher intermittent (ß 10.47, 95% CI 1.03-19.91) and constant (ß 12.62, 95% CI 3.02-22.23) pain scores. No associations were identified for synovitis in those with late radiographic OA. CONCLUSION: In patients with knee OA, moderate-to-severe synovitis is most strongly associated with constant pain. Inflammation may play context-specific roles across pain experiences, especially in earlier radiographic stages of knee OA.

Dissecting Oncogenic RTK Pathways in Colorectal Cancer Initiation and Progression.

Colorectal cancer (CRC) is a progressive disorder associated with an accumulation of multiple heterogeneous genetic alterations in intestinal epithelial cells (IEC). However, when these cells undergo neoplastic transformation and become cancerous and metastatic, they invariably acquire hallmarks conferring them the ability to hyperproliferate, escape growth-inhibitory and death-inducing cues, and promote angiogenesis as well as epithelial-to-mesenchymal transformation (EMT), fostering their invasive dissemination from primary tumor into distant tissues. Compelling clinical and experimental evidence suggest that aberrant engagement of cell surface growth factor receptor tyrosine kinase (RTK) signaling, like that of the hepatocyte growth factor (HGF)/MET receptor, underlies CRC metastatic progression by promoting these cancer hallmarks. To date, though, the use of RTK-targeting agents has been viewed as a promising approach for the treatment of metastatic CRC, clinical success has been modest.Our vision is that the prospect of designing RTK-based, improved and innovative CRC therapies and prognostic markers likely rests on a comprehensive understanding of the biological processes and underlying regulatory molecular mechanisms by which deregulation of RTK signaling governs IEC's neoplastic transformation and their transition from noninvasive to metastatic and malignant cells. Herein, we describe our scheme for defining the full scope of oncogenic MET-driven cancer biological processes, in cellulo and in vivo, as well as the individual contribution of MET-binding effectors in a nontransformed IEC model, the IEC-6 cell line.

Hypoxia-induced mobilization of NHE6 to the plasma membrane triggers endosome hyperacidification and chemoresistance.

The pH-dependent partitioning of chemotherapeutic drugs is a fundamental yet understudied drug distribution mechanism that may underlie the low success rates of current approaches to counter multidrug resistance (MDR). This mechanism is influenced by the hypoxic tumour microenvironment and results in selective trapping of weakly basic drugs into acidified compartments such as the extracellular environment. Here we report that hypoxia not only leads to acidification of the tumour microenvironment but also induces endosome hyperacidification. The acidity of the vesicular lumen, together with the alkaline pH of the cytoplasm, gives rise to a strong intracellular pH gradient that drives intravesicular drug trapping and chemoresistance. Endosome hyperacidification is due to the relocalization of the Na+/H+ exchanger isoform 6 (NHE6) from endosomes to the plasma membrane, an event that involves binding of NHE6 to the activated protein kinase C-receptor for activated C kinase 1 complex. These findings reveal a novel mechanism of hypoxia-induced MDR that involves the aberrant intracellular distribution of NHE6.

Glycogen synthase kinase-3 (GSK3) inhibition induces prosurvival autophagic signals in human pancreatic cancer cells.

Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation.

HDAC6 deacetylase activity is required for hypoxia-induced invadopodia formation and cell invasion.

Despite significant progress in the cancer field, tumor cell invasion and metastasis remain a major clinical challenge. Cell invasion across tissue boundaries depends largely on extracellular matrix degradation, which can be initiated by formation of actin-rich cell structures specialized in matrix degradation called invadopodia. Although the hypoxic microenvironment within solid tumors has been increasingly recognized as an important driver of local invasion and metastasis, little is known about how hypoxia influences invadopodia biogenesis. Here, we show that histone deacetylase 6 (HDAC6), a cytoplasmic member of the histone deacetylase family, is a novel modulator of hypoxia-induced invadopodia formation. Hypoxia was found to enhance HDAC6 tubulin deacetylase activity through activation of the EGFR pathway. Activated HDAC6, in turn, triggered Smad3 phosphorylation resulting in nuclear accumulation. Inhibition of HDAC6 activity or knockdown of the protein inhibited both hypoxia-induced Smad3 activation and invadopodia formation. Our data provide evidence that hypoxia influences invadopodia formation in a biphasic manner, which involves the activation of HDAC6 deacetylase activity by EGFR, resulting in enhanced Smad phosphorylation and nuclear accumulation. The identification of HDAC6 as a key participant of hypoxia-induced cell invasion may have important therapeutic implications for the treatment of metastasis in cancer patients.

The MEK/ERK pathway promotes NOTCH signalling in pancreatic cancer cells.

Activation of the NOTCH receptors relies on their intracellular proteolysis by the gamma-secretase complex. This cleavage liberates the NOTCH intracellular domain (NIC) thereby allowing the translocation of NIC towards the nucleus to assemble into a transcriptional platform. Little information is available regarding the regulatory steps operating on NIC following its release from the transmembrane receptor up to its association with transcriptional partners. Interfering with these regulatory steps might potentially influences the nuclear outcome of NOTCH signalling. Herein, we exploited a reliable model to study the molecular events occurring subsequent to NOTCH1 cleavage. In pancreatic cancer cells, pulse of NOTCH1 activation led to increased expression of NOTCH target genes namely HES1 and c-MYC. We uncovered that, upon its release, the NOTCH1 intracellular domain, NIC1, undergoes a series of post-translational modifications that include phosphorylation. Most interestingly, we found that activation of the MEK/ERK pathway promotes HES1 expression. Inhibition of the gamma-secretase complex prevented the MEK/ERK-induced HES1 expression suggesting a NOTCH-dependent mechanism. Finally, higher levels of NIC1 were found associated with its transcriptional partners [CBF1, Su(H) and LAG-1] (CSL) and MASTERMIND-LIKE 1 (MAML1) upon MEK/ERK activation providing a potential mechanism whereby the MEK/ERK pathway promotes expression of NOTCH target genes. For the first time, our data exposed a signalling pathway, namely the MEK/ERK pathway that positively impacts on NOTCH nuclear outcome.

Hypoxia enhances cancer cell invasion through relocalization of the proprotein convertase furin from the trans-Golgi network to the cell surface.

Tumor hypoxia is strongly associated with malignant progression such as increased cell invasion and metastasis. Although the invasion-related genes affected by hypoxia have been well described, the contribution of post-transcriptional mechanisms such as protein trafficking and proprotein processing associated with the hypoxic response remains poorly understood. The proprotein convertase furin, the major processing enzyme of the secretory pathway, resides in the trans-Golgi network and most studies support a model where endogenous substrates are processed by furin within this compartment. Here, we report that hypoxia triggered an unexpected relocalization of furin from the trans-Golgi network to endosomomal compartments and the cell surface in cancer cells. Exposing these cells back to normoxic conditions reversed furin redistribution, suggesting that the tumor microenvironment modulates furin trafficking in a highly regulated manner. Assessment of the mechanisms involved revealed that both Rab4GTPase-dependent recycling and interaction of furin with the cytoskeletal anchoring protein, filamin-A, are essential for the cell surface relocalization of furin. Interference with the association of furin with filamin-A, prevented cell surface relocalization of furin and abolished the ability of cancer cells to migrate in response to hypoxia. Our observations support the notion that hypoxia promotes the formation of a peripheral processing compartment where furin translocates for enhanced processing of proproteins involved in tumorigenesis.

Hypoxia-induced invadopodia formation involves activation of NHE-1 by the p90 ribosomal S6 kinase (p90RSK).

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na(+)/H(+) exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na(+)/H(+) exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.

Autotaxin promotes cancer invasion via the lysophosphatidic acid receptor 4: participation of the cyclic AMP/EPAC/Rac1 signaling pathway in invadopodia formation.

The ability of cancer cells to invade and metastasize is the major cause of death in cancer patients. Autotaxin (ATX) is a secreted lysophospholipase whose level of expression within tumors correlates strongly with their aggressiveness and invasiveness. ATX is the major enzyme involved in the production of lysophosphatidic acid (LPA), a phospholipid that is known to act mostly through its three first characterized receptors (LPA(1), LPA(2), and LPA(3)). Tumor cell invasion across tissue boundaries and metastasis are dependent on the capacity of invasive cancer cells to breach the basement membrane. This process can be initiated by the formation of the actin-rich cell protrusions, invadopodia. In this study, we show that ATX is implicated in the formation of invadopodia in various cancer cells types and this effect is dependent on the production of LPA. We further provide evidence that LPA(4) signaling in fibrosarcoma cells regulates invadopodia formation downstream of ATX, a process mediated through the activation of EPAC by cyclic AMP and subsequent Rac1 activation. Results using LPA(4) shRNA support the requirement of the LPA(4) receptor for cell invasion and in vivo metastasis formation. This work presents evidence that blocking the LPA receptor, LPA(4), in fibrosarcoma cells could provide an additional tool to improve the efficacy of treatment of metastasis in patients. Because LPA receptors and ATX are currently being targeted in preclinical trials, the current findings should stimulate future studies to evaluate the expression pattern and clinical outcome of LPA(4), together with other LPA receptors, in various cancer patients.

Triple vasopeptidase inhibition normalizes blood pressure in conscious, unrestrained, and spontaneously hypertensive rats.

BACKGROUND: CGS 35601 is a potent triple vasopeptidase inhibitor (VPI) of angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin-converting enzyme (ECE). The aim of the study was to determine the effects of this VPI on the hemodynamic profile of conscious, instrumented, unrestrained spontaneously hypertensive rats (SHR), in comparison to selective inhibitors of ACE and ACE + NEP, than +ECE combined. Circulating plasma concentrations of vasoactive mediators and reactive oxygen species were measured. METHODS: Old SHR male were instrumented (arterial catheter) and placed in a metabolic cage for daily hemodynamic measurements and blood samplings. Seven days after surgery, SHR received 1) saline vehicle; 2) increasing doses of the triple CGS 35601 (0.01, 0.1, 1, and 5 mg/kg/d, intra-arterially (i.a.) infusion for 5 d/dose) followed by a 5-day washout period; 3) benazepril (ACE inhibitor), ACE inhibitor + CGS 24592 (NEP inhibitor) and ACE inhibitor + NEP inhibitor + CGS 35066 (ECE inhibitor) (1 or 5 mg/kg/d i.a. infusion for 5 d/combination) followed by a 5-day washout period. RESULTS: The lowest dose of CGS 35601 had no effect. Doses at 0.1, 1, and 5 mg/kg/d reduced mean arterial blood pressure by 10%, 22%, and 40%, respectively. Heart rate was unaffected in all groups. CGS 35601 decreased concentrations of angiotensin II (Ang II), endothelin-1 (ET-1), and pro-atrial natriuretic peptide (proANP), and increased those of big ET-1, atrial natriuretic peptide (ANP), bradykinin (BK), and hydrogen peroxide (H2O2) dose dependently. CONCLUSIONS: The blood pressure-lowering effect of this triple VPI was superior to that of the other VPI in this preclinical rat model of hypertension. Further experiments are needed to assess triple VPI to other combinations in other models with regard to efficacy and angioedema. Only then it may constitute a first-in-class approach for the treatment of hypertension and other cardiovascular disorders.

Synthesis of multiwhisker-receptive fields in subcortical stations of the vibrissa system.

This study addresses the origins of multiwhisker-receptive fields of neurons in the thalamic ventral posterior medial (VPM) nucleus of the rat. We sought to determine whether multiwhisker-receptive field synthesis occurs in VPM through convergent projections from the principalis (PrV) and interpolaris (SpVi) nuclei, or in PrV by intersubnuclear projections from the spinal trigeminal complex. We tested these hypotheses by recording whisker-evoked responses in PrV and VPM before and after electrolytic lesion of the SpVi in lightly anesthetized rats. Before the lesion PrV cells responded, on average, to 3.2 +/- 1.2 whiskers but responsiveness was reduced to 1.07 +/- 0.31 whisker after the lesion. A similar reduction of receptive field size was observed in VPM, where neurons responded, on average, to 2.94 +/- 0.95 whiskers before the lesion and to 1.05 +/- 0.22 whisker after the lesion. Thus one can conclude that intersubnuclear projections mediate surround whisker-receptive fields in PrV, and therefore in VPM. However, it has previously been shown that parasagittal brain stem transection, which severed ascending projections from SpVi, but left intersubnuclear connections intact, rendered VPM cells monowhisker responsive. We wondered whether midline brain stem lesion modified receptive field properties in SpVi. In normal rats SpVi cells responded, on average, to 7.52 +/- 4.25 whiskers, but responsiveness was dramatically reduced to 1.47 +/- 1.07 whisker after the lesion. Together these results indicate that the synthesis of surround receptive fields in subcortical stations relies almost exclusively on intersubnuclear projections from the spinal trigeminal complex to the PrV.