Khalil N. Bitar

HSP27 is a mediator of sustained smooth muscle contraction in response to bombesin

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.

Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27

The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60(src), respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10(-7) M) or ceramide (10(-7) M) ( approximately 30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60(src) inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60(src) and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60 src , respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10-7 M) or ceramide (10-7 M) (∼30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60 src inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60 src and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.

HSP27 in signal transduction and association with contractile proteins in smooth muscle cells.

Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that 1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide; 2) PKC-α and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-α translocation is inhibited by VIP; 3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.

Regenerative Medicine as Applied to General Surgery

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

Successful Implantation of Bioengineered, Intrinsically Innervated, Human Internal Anal Sphincter

BACKGROUND & AIMS To restore fecal continence, the weakened pressure of the internal anal sphincter (IAS) must be increased. We bioengineered intrinsically innervated human IAS to emulate sphincteric physiology in vitro. METHODS We cocultured human IAS circular smooth muscle with immortomouse fetal enteric neurons. We investigated the ability of bioengineered innervated human IAS, implanted in RAG1-/- mice, to undergo neovascularization and preserve the physiology of the constituent myogenic and neuronal components. RESULTS The implanted IAS was neovascularized in vivo; numerous blood vessels were observed with no signs of inflammation or infection. Real-time force acquisition from implanted and preimplant IAS showed distinct characteristics of IAS physiology. Features included the development of spontaneous myogenic basal tone; relaxation of 100% of basal tone in response to inhibitory neurotransmitter vasoactive intestinal peptide (VIP) and direct electrical field stimulation of the intrinsic innervation; inhibition of nitrergic and VIPergic electrical field-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction); contraction in response to cholinergic stimulation with acetylcholine; and intact electromechanical coupling (evidenced by direct response to potassium chloride). Implanted, intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiology of myogenic and neuronal components. CONCLUSIONS Intrinsically innervated human IAS bioengineered tissue can be successfully implanted in mice. This approach might be used to treat patients with fecal incontinence.

Bioengineered Internal Anal Sphincter Derived From Isolated Human Internal Anal Sphincter Smooth Muscle Cells

BACKGROUND & AIMS The internal anal sphincter (IAS) is a specialized circular smooth muscle that maintains rectoanal continence. In vitro models are needed to study the pathophysiology of human IAS disorders. We bioengineered sphincteric rings from human IAS smooth muscle cells (SMC) and investigated their response to cholinergic stimulation as well as investigated whether protein kinase C (PKC) and Rho kinase signaling pathways remain functional. METHODS 3-Dimensional bioengineered ring (3DBR) model of the human IAS was constructed from isolated human IAS SMC obtained from surgery. Contractile properties and force generation in response to acetylcholine, PKC inhibitor calphostin-C, Rho/ROCK inhibitor Y-27632, permeable Rho/ROCK inhibitor c3-exoenzyme, and PKC activator PdBU was measured. RESULTS The human IAS 3DBR has the essential characteristics of physiologically functional IAS; it generated a spontaneous myogenic basal tone, and the constructs were able to relax in response to relaxants and contract in response to contractile agents. The constructs generated dose-dependent force in response to acetylcholine. Basal tone was significantly reduced by calphostin-C but not with Y-27632. Acetylcholine-induced force generation was also significantly reduced by calphostin-C but not with Y-27632. PdBU generated force that was equal in magnitude to acetylcholine. Thus, calphostin-C inhibited PdBU-induced force generation, whereas Y-27632 and c3 exoenzyme did not. CONCLUSIONS These data indicate that basal tone and acetylcholine-induced force generation depend on signaling through the PKC pathway in human IAS; PKC-mediated force generation is independent of the Rho/ROCK pathway. This human IAS 3DBR model can be used to study the pathophysiology associated with IAS malfunctions.

IGF-I induces collagen and IGFBP-5 mRNA in rat intestinal smooth muscle

Insulin-like growth factor (IGF) binding protein 5 (IGFBP-5) mRNA was studied in intestines of rats with peptidoglycan-polysaccharide enterocolitis by Northern analysis and in situ hybridization. IGFBP-5 mRNA was increased 2.4 ± 0.5-fold in inflamed rat colon compared with controls and was highly expressed in smooth muscle. Cultured rat intestinal smooth muscle cells were used to study the regulation of IGFBP-5 and type I collagen synthesis. IGF-I (100 ng/ml) increased IGFBP-5 mRNA (1.9 ± 0.1-fold) and collagen type α1(I) mRNA (1.6 ± 0.2-fold) in cultured smooth muscle cells. IGF-I induced a dose- and time-dependent increase in IGFBP-5 in conditioned medium by Western ligand blot and by immunoblot. IGF-I did not affect the IGFBP-5 mRNA decay rate after transcriptional blockade. Cycloheximide abolished IGFBP-5 mRNA. In conclusion, IGFBP-5 mRNA is expressed by intestinal smooth muscle and is increased during chronic inflammation. IGF-I increases IGFBP-5 and collagen mRNAs in intestinal smooth muscle cells.

Inhibition of Glycogen Synthase Kinase-3β Is Sufficient for Airway Smooth Muscle Hypertrophy

We examined the role of glycogen synthase kinase-3β (GSK-3β) inhibition in airway smooth muscle hypertrophy, a structural change found in patients with severe asthma. LiCl, SB216763, and specific small interfering RNA (siRNA) against GSK-3β, each of which inhibit GSK-3β activity or expression, increased human bronchial smooth muscle cell size, protein synthesis, and expression of the contractile proteins α-smooth muscle actin, myosin light chain kinase, smooth muscle myosin heavy chain, and SM22. Similar results were obtained following treatment of cells with cardiotrophin (CT)-1, a member of the interleukin-6 superfamily, and transforming growth factor (TGF)-β, a proasthmatic cytokine. GSK-3β inhibition increased mRNA expression of α-actin and transactivation of nuclear factors of activated T cells and serum response factor. siRNA against eukaryotic translation initiation factor 2Bϵ (eIF2Bϵ) attenuated LiCl- and SB216763-induced protein synthesis and expression of α-actin and SM22, indicating that eIF2B is required for GSK-3β-mediated airway smooth muscle hypertrophy. eIF2Bϵ siRNA also blocked CT-1- but not TGF-β-induced protein synthesis. Infection of human bronchial smooth muscle cells with pMSCV GSK-3β-A9, a retroviral vector encoding a constitutively active, nonphosphorylatable GSK-3β, blocked protein synthesis and α-actin expression induced by LiCl, SB216763, and CT-1 but not TGF-β. Finally, lungs from ovalbumin-sensitized and -challenged mice demonstrated increased α-actin and CT-1 mRNA expression, and airway myocytes isolated from ovalbumin-treated mice showed increased cell size and GSK-3β phosphorylation. These data suggest that inhibition of the GSK-3β/eIF2Bϵ translational control pathway contributes to airway smooth muscle hypertrophy in vitro and in vivo. On the other hand, TGF-β-induced hypertrophy does not depend on GSK-3β/eIF2B signaling.

Airway smooth muscle hyperplasia and hypertrophy correlate with glycogen synthase kinase-3β phosphorylation in a mouse model of asthma

Increased airway smooth muscle (ASM) mass, a characteristic finding in asthma, may be caused by hyperplasia or hypertrophy. Cell growth requires increased translation of contractile apparatus mRNA, which is controlled, in part, by glycogen synthase kinase (GSK)-3beta, a constitutively active kinase that inhibits eukaryotic initiation factor-2 activity and binding of methionyl tRNA to the ribosome. Phosphorylation of GSK-3beta inactivates it, enhancing translation. We sought to quantify the contributions of hyperplasia and hypertrophy to increased ASM mass in ovalbumin (OVA)-sensitized and -challenged BALB/c mice and the role of GSK-3beta in this process. Immunofluorescent probes, confocal microscopy, and stereological methods were used to analyze the number and volume of cells expressing alpha-smooth muscle actin and phospho-Ser(9) GSK-3beta (pGSK). OVA treatment caused a 3-fold increase in ASM fractional unit volume or volume density (Vv) (PBS, 0.006 +/- 0.0003; OVA, 0.014 +/- 0.001), a 1.5-fold increase in ASM number per unit volume (Nv), and a 59% increase in volume per cell (Vv/Nv) (PBS, 824 +/- 76 microm(3); OVA, 1,310 +/- 183 mum(3)). In OVA-treated mice, there was a 12-fold increase in the Vv of pGSK (+) ASM, a 5-fold increase in the Nv of pGSK (+) ASM, and a 1.6-fold increase in Vv/Nv. Lung homogenates from OVA-treated mice showed increased GSK-3beta phosphorylation and lower GSK-3beta activity. Both hyperplasia and hypertrophy are responsible for increased ASM mass in OVA-treated mice. Phosphorylation and inactivation of GSK-3beta are associated with ASM hypertrophy, suggesting that this kinase may play a role in asthmatic airway remodeling.

Aging and gastrointestinal smooth muscle

The present review is an attempt to put into perspective the available information on the putative changes in cellular mechanisms of the contractile properties of the aging gastrointestinal (GI) smooth muscle. Information on smooth muscle of the GI tract is scanty. Smooth muscle cells from old rats (32 months old) exhibit limited cell length distribution and diminished contractility. The observed reduced contractile response may be due to the effect of aging on signal transduction pathways, especially an inhibition of the tyrosine kinase-Src kinase pathway, a reduced activation of the PKCalpha pathway, a reduced association of contractile proteins (HSP27-tropomyosin, HSP27-actin, and actin-myosin). Levels of HSP27-phosphorylation are also reduced compared to adult rats. Regulation of GI motility is a complex mechanism of signal transduction and interaction of signaling and contractile proteins. It is suggested that further studies to elucidate the role of HSP27 in aging smooth muscle of the GI tract are needed.