Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.

Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.

The potential anti-inflammatory and anti-nociceptive effects of rat hemopressin (PVNFKFLSH) in experimental arthritis.

Inflammatory arthritis, such as rheumatoid arthritis (RA), stands out as one of the main sources of pain and impairment to the quality of life. The use of hemopressin (PVNFKFLSH; Hp), an inverse agonist of type 1 cannabinoid receptor, has proven to be effective in producing analgesia in pain models, but its effect on neuro-inflammatory aspects of RA is limited. In this study, antigen-induced arthritis (AIA) was evoked by the intraarticular (i.art.) injection of methylated bovine serum albumin (mBSA) in male Sprague Dawley rats. Phosphate buffered saline (PBS)-injected ipsilateral knee joints or AIA contralateral were used as control. Nociceptive and inflammatory parameters such as knee joint oedema and leukocyte influx and histopathological changes were carried out in addition to the local measurement of interleukins (IL) IL-6, IL-1ß, tumor necrosis factor-α and the immunoreactivity of the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) in the spinal cord (lumbar L3-5 segments) of AIA rats. For 4 days, AIA rats were treated daily with a single administration of saline, Hp injected (10 or 20 µg/day, i.art.), Hp given orally (20 µg/Kg, p.o.) or indomethacin (Indo; 5 mg/Kg, i.p.). In comparison to the PBS control group, the induction of AIA produced a significant and progressive mono-arthritis condition. The degree of AIA severity progressively compromised the normal walking pattern and impaired mobility over the next four days in relation to PBS-injected rats or contralateral knee joints. In AIA rats, the reduction of the distance between footprints and disturbances of gait evidenced signs of nociception. This response worsened at day 4, and a loss of footprint from the ipsilateral hind paw was evident. Daily treatment of the animals with Hp either i.art. (10 and 20 µg/knee) or p.o. (20 µg/Kg) as well as Indo (5 mg/Kg, i.p.) ameliorated the impaired mobility in a time-dependent manner (P < 0.05). In parallel, the AIA-injected ipsilateral knee joints reach a peak of swelling 24 h after AIA induction, which persisted over the next four days in relation to PBS-injected rats or contralateral knee joints. There was a significant but not dose-dependent inhibitory effect produced by all dosages and routes of Hp treatments on AIA-induced knee joint swelling (P < 0.05). In addition, the increased synovial levels of MPO activity, total leukocytes number and IL-6, but not IL-1ß, were significantly reduced by the lower i.art. dose of Hp. In conclusion, these results successfully demonstrate that Hp may represent a novel therapeutic strategy to treat RA, an effect which is unrelated to the proinflammatory actions of the neuropeptides CGRP and SP.

Tumor necrosis factor receptor-associated factor 6 interaction with alpha-synuclein enhances cell death through the Nuclear Factor-kB pathway.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease characterized by intracellular inclusions named Lewy bodies (LB), and alpha-synuclein (asyn) is the major component of these protein aggregates. The precise physiological and pathological roles of asyn are not fully understood. Nevertheless, asyn present in LB is ubiquitinated but fails to reach the 26S proteasome. The mutation A30 P is related to an aggressive and early-onset form of PD. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase, and it interacts and ubiquitinates the asyn in atypical chains (lysine K6, K27, K29, and K33). Methods: Here, we investigated the role of TRAF6 interaction with asyn and the involvement of nuclear factor κB (NF-κB), a key transcription factor in pro-inflammatory signaling pathway activation. RESULTS AND CONCLUSION: We demonstrated that TRAF6 binds to both WT and the mutant form A30 P asyn in an SH-SY5Y cell model. Additionally, the interaction between TRAF6 and WT asyn induced an increase in the activation of NF-κB, leading to changes in TNF, IL-1ß and IL-10 levels and culminating in reduced cell viability. Interestingly, the activation of NF-κB and gene regulation were not found in A30 P asyn. These data point to a novel role of TRAF6 in the pathophysiology of PD.

The potential anti-inflammatory and anti-nociceptive effects of rat hemopressin (PVNFKFLSH) in experimental arthritis

Inflammatory arthritis, such as rheumatoid arthritis (RA), stands out as one of the main sources of pain and impairment to the quality of life. The use of hemopressin (PVNFKFLSH; Hp), an inverse agonist of type 1 cannabinoid receptor, has proven to be effective in producing analgesia in pain models, but its effect on neuro-inflammatory aspects of RA is limited. In this study, antigen-induced arthritis (AIA) was evoked by the intraarticular (i.art.) injection of methylated bovine serum albumin (mBSA) in male Sprague Dawley rats. Phosphate buffered saline (PBS)-injected ipsilateral knee joints or AIA contralateral were used as control. Nociceptive and inflammatory parameters such as knee joint oedema and leukocyte influx and histopathological changes were carried out in addition to the local measurement of interleukins (IL) IL-6, IL-1β, tumor necrosis factor-α and the immunoreactivity of the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) in the spinal cord (lumbar L3-5 segments) of AIA rats. For 4 days, AIA rats were treated daily with a single administration of saline, Hp injected (10 or 20 μg/day, i.art.), Hp given orally (20 μg/Kg, p.o.) or indomethacin (Indo; 5 mg/Kg, i.p.). In comparison to the PBS control group, the induction of AIA produced a significant and progressive mono-arthritis condition. The degree of AIA severity progressively compromised the normal walking pattern and impaired mobility over the next four days in relation to PBS-injected rats or contralateral knee joints. In AIA rats, the reduction of the distance between footprints and disturbances of gait evidenced signs of nociception. This response worsened at day 4, and a loss of footprint from the ipsilateral hind paw was evident. Daily treatment of the animals with Hp either i.art. (10 and 20 μg/knee) or p.o. (20 μg/Kg) as well as Indo (5 mg/Kg, i.p.) ameliorated the impaired mobility in a time-dependent manner (P < 0.05). In parallel, the AIA-injected ipsilateral knee joints reach a peak of swelling 24 h after AIA induction, which persisted over the next four days in relation to PBS-injected rats or contralateral knee joints. There was a significant but not dose-dependent inhibitory effect produced by all dosages and routes of Hp treatments on AIA-induced knee joint swelling (P < 0.05). In addition, the increased synovial levels of MPO activity, total leukocytes number and IL-6, but not IL-1β, were significantly reduced by the lower i.art. dose of Hp. In conclusion, these results successfully demonstrate that Hp may represent a novel therapeutic strategy to treat RA, an effect which is unrelated to the proinflammatory actions of the neuropeptides CGRP and SP

Effect of tryptase inhibition on joint inflammation: a pharmacological and lentivirus-mediated gene transfer study.

BACKGROUND: Increasing evidences indicate that an unbalance between tryptases and their endogenous inhibitors, leading to an increased proteolytic activity, is implicated in the pathophysiology of rheumatoid arthritis. The aim of the present study was to evaluate the impact of tryptase inhibition on experimental arthritis. METHODS: Analysis of gene expression and regulation in the mouse knee joint was performed by RT-qPCR and in situ hybridization. Arthritis was induced in male C57BL/6 mice with mBSA/IL-1ß. Tryptase was inhibited by two approaches: a lentivirus-mediated heterologous expression of the human endogenous tryptase inhibitor, sperm-associated antigen 11B isoform C (hSPAG11B/C), or a chronic treatment with the synthetic tryptase inhibitor APC366. Several inflammatory parameters were evaluated, such as oedema formation, histopathology, production of IL-1ß, -6, -17A and CXCL1/KC, myeloperoxidase and tryptase-like activities. RESULTS: Spag11c was constitutively expressed in chondrocytes and cells from the synovial membrane in mice, but its expression did not change 7 days after the induction of arthritis, while tryptase expression and activity were upregulated. The intra-articular transduction of animals with the lentivirus phSPAG11B/C or the treatment with APC366 inhibited the increase of tryptase-like activity, the late phase of oedema formation, the production of IL-6 and CXCL1/KC. In contrast, neutrophil infiltration, degeneration of hyaline cartilage and erosion of subchondral bone were not affected. CONCLUSIONS: Tryptase inhibition was effective in inhibiting some inflammatory parameters associated to mBSA/IL-1ß-induced arthritis, notably late phase oedema formation and IL-6 production, but not neutrophil infiltration and joint degeneration. These results suggest that the therapeutic application of tryptase inhibitors to rheumatoid arthritis would be restrained to palliative care, but not as disease-modifying drugs. Finally, this study highlighted lentivirus-based gene delivery as an instrumental tool to study the relevance of target genes in synovial joint physiology and disease.

Suppression of MAPK attenuates neuronal cell death induced by activated glia-conditioned medium in alpha-synuclein overexpressing SH-SY5Y cells.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease with characteristics and symptoms that are well defined. Nevertheless, its aetiology remains unknown. PD is characterized by the presence of Lewy bodies inside neurons. α-Synuclein (α-syn) is a soluble protein present in the pre-synaptic terminal of neurons. Evidence suggests that α-syn has a fundamental role in PD pathogenesis, given that it is an important component of Lewy bodies localized in the dopaminergic neurons of PD patients. METHODS: In the present study, we investigated the influence of wild type (WT) and A30P α-syn overexpression on neuroblastoma SH-SY5Y toxicity induced by the conditioned medium (CM) from primary cultures of glia challenged with lipopolysaccharide (LPS) from Escherichia coli. RESULTS: We observed that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show evidence of cell death, which is not reverted by NF-κB inhibition by sodium salicylate or by blockage of P50 (NF-κB subunit). Furthermore, the expression of A30P α-syn in neuroblastoma SH-SY5Y decreases the cell death triggered by the CM of activated glia versus WT α-syn or control group. This effect of A30P α-syn may be due to the low MAPK42/44 phosphorylation. This finding is substantiated by MEK1 inhibition by PD98059, decreasing LDH release by CM in SH-SY5Y cells. CONCLUSION: Our results suggest that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show cell death, which is not reverted by NF-κB blockage. Additionally, the expression of A30P α-syn on neuroblastoma SH-SY5Y leads to decreased cell death triggered by the CM of activated glia, when compared to WT α-syn or control group. The mechanism underlying this process remains to be completely elucidated, but the present data suggest that MAPK42/44 phosphorylation plays an important role in this process. PROSPERO: CRD42015020829.

Peripheral neurokinin-1 receptors contribute to kaolin-induced acute monoarthritis in rats.

OBJECTIVE: intra-articular co-injection of kaolin with carrageenan (CGN) in rodents is widely used as an experimental model of arthritis. However, the ability of kaolin to cause arthritis and related immune responses when administered alone is unclear. We evaluated the contribution of prostanoids and sensory C-fibres (and their neuropeptide substance P) to kaolin-induced inflammation in the rat knee. METHODS: Wistar rats, 8-10 weeks old, received an intra-articular injection of kaolin (1-10 µg/joint) or saline into the knee joint. Knee inflammation, proinflammatory cytokines, pain behaviour and secondary tactile allodynia were assessed over 5 h, when synovial leukocyte counts, histopathological changes and proinflammatory cytokine levels were evaluated. RESULTS: The intra-articular injection of kaolin caused a dose- and time-dependent knee swelling and impairment of motion that were associated with secondary tactile allodynia, elevated concentrations of IL-1ß, IL-6 and TNFα, leukocyte infiltration, and histopathological changes in the ipsilateral hindpaw. The neurokinin-1 (NK1) receptor antagonist SR140333 or neonatal treatment with capsaicin markedly reduced the inflammatory parameters, cytokines and allodynia but failed to significantly inhibit the impaired motion. The cyclo-oxygenase inhibitor indomethacin partially inhibited knee oedema and allodynia but did not affect the leukocyte influx, myeloperoxidase activity or impaired motion in the kaolin-injected rat. CONCLUSIONS: We show the first evidence that intra-articular injection of kaolin without CGN produced severe acute monoarthritis. This was highly dependent on substance P (released from C-fibres) and NK1 receptor activation, which stimulated local production of proinflammatory cytokines. This model may be of critical importance for mechanistic studies and screening new anti-inflammatory/analgesic drugs.

Dynamic changes in the spatio-temporal expression of the ß-defensin SPAG11C in the developing rat epididymis and its regulation by androgens.

Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the ß-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of ß-defensins in the epididymis.

Role of transient receptor potential vanilloid 4 in rat joint inflammation.

OBJECTIVE: To determine whether activation of transient receptor potential vanilloid 4 (TRPV-4) induces inflammation in the rat temporomandibular joint (TMJ), and to assess the effects of TRPV-4 agonists and proinflammatory mediators, such as a protease-activated receptor 2 (PAR-2) agonist, on TRPV-4 responses. METHODS: Four hours after intraarticular injection of carrageenan into the rat joints, expression of TRPV-4 and PAR-2 in trigeminal ganglion (TG) neurons and in the TMJs were evaluated by real-time reverse transcription-polymerase chain reaction and immunofluorescence, followed by confocal microscopy. The functionality of TRPV-4 and its sensitization by a PAR-2-activating peptide (PAR-2-AP) were analyzed by measuring the intracellular Ca(2+) concentration in TMJ fibroblast-like synovial cells or TG neurons. Plasma extravasation, myeloperoxidase activity, and the head-withdrawal threshold (index of mechanical allodynia) were evaluated after intraarticular injection of selective TRPV-4 agonists, either injected alone or coinjected with PAR-2-AP. RESULTS: In the rat TMJs, TRPV-4 and PAR-2 expression levels were up-regulated after the induction of inflammation. Two TRPV-4 agonists specifically activated calcium influx in TMJ fibroblast-like synovial cells or TG neurons. In vivo, the agonists triggered dose-dependent increases in plasma extravasation, myeloperoxidase activity, and mechanical allodynia. In synovial cells or TG neurons, pretreatment with PAR-2-AP potentiated a TRPV-4 agonist-induced increase in [Ca(2+) ](i) . In addition, TRPV-4 agonist-induced inflammation was potentiated by PAR-2-AP in vivo. CONCLUSION: In this rat model, TRPV-4 is expressed and functional in TG neurons and synovial cells, and activation of TRPV-4 in vivo causes inflammation in the TMJ. Proinflammatory mediators, such as PAR-2 agonists, sensitize the activity of TRPV-4. These results identify TRPV-4 as an important signal of inflammation in the joint.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint.

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RT-PCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3+/-49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment.

Participation of peripheral tachykinin NK1 receptors in the carrageenan-induced inflammation of the rat temporomandibular joint.

Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK(1) receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNFalpha and IL-1beta concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK(1) receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24h, as well as the production of TNFalpha and IL-1beta into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK(1) receptors mediate the production of both TNFalpha and IL-1beta in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component.