Targeting Vascular Endothelial Growth Factor Receptors as a Therapeutic Strategy for Osteoarthritis and Associated Pain.

Pain is the major reason that patients suffering from osteoarthritis (OA) seek medical care. We found that vascular endothelial growth factors (VEGFs) mediate signaling in OA pain pathways. To determine the specific contributions of VEGFs and their receptors (VEGFRs) to joint pathology and pain transmission during OA progression, we studied intra-articular (IA) injections of VEGF ligands into murine knee joints. Only VEGF ligands specific for the activation of VEGFR1, but not VEGFR2, induced allodynia within 30 min. Interventions in OA by inhibitors of VEGFRs were done in vivo using a preclinical murine OA model by IA injections of selective inhibitors of VEGFR1/VEGFR2 kinase (pazopanib) or VEGFR2 kinase (vandetanib). OA phenotypes were evaluated using pain-associated murine behavioral tests and histopathologic analyses. Alterations in VEGF/VEGFR signaling by drugs were determined in knee joints, dorsal root ganglia, and spinal cord by immunofluorescence microscopy. Pazopanib immediately relieved OA pain by interfering with pain transmission pathways. Pain reduction by vandetanib was mainly due to the inhibition of cartilage degeneration by suppressing VEGFR2 expression. In conclusion, IA administration of pazopanib, which simultaneously inhibits VEGFR1 and VEGFR2, can be developed as an ideal OA disease-modifying drug that rapidly reduces joint pain and simultaneously inhibits cartilage degeneration.

NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

Absence of VEGFR-1/Flt-1 signaling pathway in mice results in insensitivity to discogenic low back pain in an established disc injury mouse model.

Although degenerative disc disease (DDD) and related low back pain (LBP) are growing public health problems, the underlying disease mechanisms remain unclear. An increase in the vascular endothelial growth factor (VEGF) levels in DDD has been reported. This study aimed to examine the role of VEGF receptors (VEGFRs) in DDD, using a mouse model of DDD. Progressive DDD was induced by anterior stabbing of lumbar intervertebral discs in wild type (WT) and VEGFR-1 tyrosine-kinase deficient mice (vegfr-1TK-/- ). Pain assessments were performed weekly for 12 weeks. Histological and immunohistochemical assessments were made for discs, dorsal root ganglions, and spinal cord. Both vegfr-1TK-/- and WT mice presented with similar pathological changes in discs with an increased expression of inflammatory cytokines and matrix-degrading enzymes. Despite the similar pathological patterns, vegfr-1TK-/- mice showed insensitivity to pain compared with WT mice. This insensitivity to discogenic pain was related to lower levels of pain factors in the discs and peripheral sensory neurons and lower spinal glial activation in the vegfr-1TK- /- mice than in the WT mice. Exogenous stimulation of bovine disc cells with VEGF increased inflammatory and cartilage degrading enzyme. Silencing vegfr-1 by small-interfering-RNA decreased VEGF-induced expression of pain markers, while silencing vegfr-2 decreased VEGF-induced expression of inflammatory and metabolic markers without changing pain markers. This suggests the involvement of VEGFR-1 signaling specifically in pain transmission. Collectively, our results indicate that the VEGF signaling is involved in DDD. Particularly, VEGFR-1 is critical for discogenic LBP transmission independent of the degree of disc pathology.

Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon.

Keratins (K) are intermediate filament proteins important in protection from stress. The roles of keratins in the intestine are not clear, but K8 knockout (K8(-/-)) mice develop a Th2-type colonic inflammation, epithelial hyperproliferation, and mild diarrhea caused by a keratin level-dependent decrease in short-circuit current and net sodium and chloride absorption in the distal colon. The lack of K8 leads to mistargeting or altered levels of membrane proteins in colonocytes; however, the main transporter responsible for the keratin-related ion transport defect is unknown. We here analyzed protein and mRNA levels of candidate ion transporters CFTR, PAT-1, NHE-3, and DRA in ileum, cecum, and proximal and distal colon. Although no differences were observed for CFTR, PAT-1, or NHE-3, DRA mRNA levels were decreased by three- to fourfold and DRA protein was almost entirely lost in K8(-/-) cecum and proximal and distal colon compared with K8(+/+), whereas the levels in ileum were normal. In K8(+/-) mice, DRA mRNA levels were unaltered, while decreased DRA protein levels were detected in the proximal colon. Immunofluorescence staining confirmed the loss of DRA in K8(-/-) distal colon, while K8(+/-) displayed a similar but more patchy apical DRA distribution compared with K8(+/+) DRA was similarly decreased when K8 was knocked down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammation-independent manner. Taken together, the loss of DRA in the K8(-/-) mouse colon and cecum explains the dramatic chloride transport defect and diarrheal phenotype after K8 inactivation and identifies K8 as a novel regulator of DRA.

Lactobacillus acidophilus alleviates platelet-activating factor-induced inflammatory responses in human intestinal epithelial cells.

Probiotics have been used as alternative prevention and therapy modalities in intestinal inflammatory disorders including inflammatory bowel diseases (IBD) and necrotizing enterocolitis (NEC). Pathophysiology of IBD and NEC includes the production of diverse lipid mediators, including platelet-activating factor (PAF) that mediate inflammatory responses in the disease. PAF is known to activate NF-κB, however, the mechanisms of PAF-induced inflammation are not fully defined. We have recently described a novel PAF-triggered pathway of NF-κB activation and IL-8 production in intestinal epithelial cells (IECs), requiring the pivotal role of the adaptor protein Bcl10 and its interactions with CARMA3 and MALT1. The current studies examined the potential role of the probiotic Lactobacillus acidophilus in reversing the PAF-induced, Bcl10-dependent NF-κB activation and IL-8 production in IECs. PAF treatment (5 µM×24 h) of NCM460 and Caco-2 cells significantly increased nuclear p65 NF-κB levels and IL-8 secretion (2-3-fold, P<0.05), compared to control, which were blocked by pretreatment of the cells for 6 h with L. acidophilus (LA) or its culture supernatant (CS), followed by continued treatments with PAF for 24 h. LA-CS also attenuated PAF-induced increase in Bcl10 mRNA and protein levels and Bcl10 promoter activity. LA-CS did not alter PAF-induced interaction of Bcl10 with CARMA3, but attenuated Bcl10 interaction with MALT1 and also PAF-induced ubiquitination of IKKγ. Efficacy of bacteria-free CS of LA in counteracting PAF-induced inflammatory cascade suggests that soluble factor(s) in the CS of LA mediate these effects. These results define a novel mechanism by which probiotics counteract PAF-induced inflammation in IECs.

Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells.

Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether KGF2 directly regulates Pgp in the human intestine is not known. Therefore, the present studies were undertaken to determine the modulation of Pgp by KGF2 using Caco-2 cells. Short-term treatment of Caco-2 cells with KGF2 (10 ng/ml, 1 h) increased Pgp activity (~2-fold, P < 0.05) as measured by verapamil-sensitive [(3)H]digoxin flux. This increase in Pgp function was associated with an increase in surface Pgp levels. The specific fibroblast growth factor receptor (FGFR) antagonist PD-161570 blocked the KGF2-mediated increase in Pgp activity. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 attenuated the stimulatory effects of KGF2 on Pgp activity. Small-interfering RNA knockdown of Erk1/2 MAPK blocked the increase in surface Pgp levels by KGF2. Long-term treatment with KGF2 (10 ng/ml, 24 h) also significantly increased PgP activity, mRNA, protein expression, and promoter activity. The long-term effects of KGF2 on Pgp promoter activity were also blocked by the FGFR antagonist and mediated by the Erk1/2 MAPK pathway. In conclusion, our findings define the posttranslational and transcriptional mechanisms underlying stimulation of Pgp function and expression by KGF2 that may contribute to the beneficial effects of KGF2 in intestinal inflammatory disorders.

Epidermal growth factor upregulates serotonin transporter in human intestinal epithelial cells via transcriptional mechanisms.

Serotonin transporter (SERT) regulates extracellular availability of serotonin and is a potential pharmacological target for gastrointestinal disorders. A decrease in SERT has been implicated in intestinal inflammatory and diarrheal disorders. However, little is known regarding regulation of SERT in the intestine. Epidermal growth factor (EGF) is known to influence intestinal electrolyte and nutrient transport processes and has protective effects on intestinal mucosa. Whether EGF regulates SERT in the human intestine is not known. The present studies examined the regulation of SERT by EGF, utilizing Caco-2 cells grown on Transwell inserts as an in vitro model. Treatment with EGF from the basolateral side (10 ng/ml, 24 h) significantly stimulated SERT activity (∼2-fold, P < 0.01) and mRNA levels compared with control. EGF increased the activities of the two alternate promoter constructs for human SERT gene: SERT promoter 1 (hSERTp1, upstream of exon 1a) and SERT promoter 2 (hSERTp2, upstream of exon 2). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1 nM) blocked the stimulatory effects of EGF on SERT promoters. Progressive deletions of the SERT promoter indicated that the putative EGF-responsive elements are present in the -672/-472 region of the hSERTp1 and regions spanning -1195/-738 and -152/+123 of hSERTp2. EGF markedly increased the binding of Caco-2 nuclear proteins to the potential AP-1 cis-elements present in EGF-responsive regions of hSERTp1 and p2. Overexpression of c-jun but not c-fos specifically transactivated hSERTp2, with no effects on hSERTp1. Our findings define novel mechanisms of transcriptional regulation of SERT by EGF via EGFR at the promoter level that may contribute to the beneficial effects of EGF in gut disorders.