Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF-Rß.

Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet-derived growth factor (PDGF)-BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF-Rß and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF-BB. CSMC were enzymatically isolated from Sprague-Dawley rats, and the effect of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, transforming growth factor (TGF), IL-17A and IL-2 on CSMC growth and responsiveness to PDGF-BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid-induced colitis. Neither CM alone nor cytokines caused proliferation of early-passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF-BB. IL-1ß, TNF-α and IL-17A, but not other cytokines, increased the effect of PDGF-BB because of up-regulation of mRNA and protein for PDGF-Rß without change in receptor phosphorylation. PDGF-BB was identified in adult rat serum (RS) and RS-induced CSMC proliferation was inhibited by imatinib, suggesting that blood-derived PDGF-BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL-1ß and TNF-α induced the early expression of PDGF-Rß, while imatinib blocked subsequent RS-induced cell proliferation. Thus, pro-inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF-Rß and serum-derived PDGF-BB, and control of PDGF-Rß expression may be beneficial in chronic intestinal inflammation.

Neuronal nitric oxide inhibits intestinal smooth muscle growth.

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [(3)H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [(3)H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo.

Discrete responses of myenteric neurons to structural and functional damage by neurotoxins in vitro.

Damage to the enteric nervous system is implicated in human disease and animal models of inflammatory bowel disease, diabetes, and Parkinson's disease, but the mechanism of death and the response of surviving neurons are poorly understood. We explored this in a coculture model of myenteric neurons, glia, and smooth muscle during exposure to the established or potential neurotoxins botulinum A, hydrogen peroxide, and acrylamide. Neuronal survival, axonal degeneration and regeneration, and neurotransmitter release were assessed during acute exposure (0-24 h) to neurotoxin and subsequent recovery (96-144 h). Unique and selective responses to each neurotoxin were found with acrylamide (0.5-2.0 mM) causing a 30% decrease in axon number without neuronal loss, whereas hydrogen peroxide (1-200 microM) caused a parallel loss in both axon and neuron number. Immunoblotting identified the loss of synaptic vesicle proteins that paralleled axon damage and was associated with marked suppression of depolarization-induced release of acetylcholine (ACh). The caspase inhibitor zVAD, but not DEVD, significantly prevented neuronal death, implying a largely caspase-3/7-independent mechanism of apoptotic death that was supported by staining for annexin V and cleaved caspase-3. In contrast, botulinum A (2 microg/ml) caused a 40% decrease in ACh release without effect on neuronal survival or axon structure. By 96 h after exposure to acrylamide or hydrogen peroxide, axon number was restored to or even surpassed the level of time-matched controls, regardless of partial neuronal loss, but ACh release remained markedly suppressed. Neural responses to toxic factors are initially unique but then converge upon robust axonal regeneration, whereas neurotransmitter release is both vulnerable to damage and slow to recover.