Robert R. Gilmont

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.

Endothelial barrier dysfunction caused by LPS correlates with phosphorylation of HSP27 in vivo

Lung edema during sepsis is triggered by formation of gaps between endothelial cells followed by macrophage infiltration. Endothelial gap formation has been proposed to involve changes in the structure of the actin filament cytoskeleton. Heat shock protein 27 (HSP27) is believed to modulate actin filament dynamics or structure, in a manner dependent on its phosphorylation status. We hypothesized that HSP27 may play a role in endothelial gap formation, by affecting actin dependent events in endothelial cells. As there has been no report concerning HSP27 in lung edemain vivo, we examined induction and phosphorylation of HSP27 in lung following LPS injection, as a model of sepsis. In lung, HSP27 mainly localized in capillary endothelial cells of the alveolus, and in smooth muscle cells of pulmonary arteries. HSP27 became significantly more phosphorylated at 3 h after LPS treatment, while the distribution of HSP27 remained unchanged. Pre-treatment with anti-TNFα antibody, which has been shown to reduce lung injury, blocked increases in HSP27 phosphorylation at 3 h. HSP27 phosphorylation was also increased in cultured rat pulmonary arterial endothelial cells (RPAEC) by treatment with TNFα, LPS, or H2O2. This phosphorylation was blocked by pre-treatment with SB203580, an inhibitor of the upstream kinase, p38 MAP kinase. Increased endothelial permeability caused by H2O2in vitro was also blocked by SB203580. The amount of actin associated with HSP27 was reduced after treatment with LPS, or H2O2. In summary, HSP27 phosphorylation temporally correlated with LPS induced pathological endothelial cell gap formationin vivo and in a cell culture model system. This is the first report of increased HSP27 phosphorylation associated with pathological lung injury in an animal model of sepsis.

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.

HSP27 regulates fibroblast adhesion, motility, and matrix contraction

Abstract Heat shock protein 27 (HSP27) modulates actin-dependent cell functions in several systems. We hypothesized that HSP27 modulates wound contraction. Stably transfected fibroblast cell lines that overexpress HSP27 (SS12) or underexpress HSP27 (AS10) were established, and cell behaviors related to wound contraction were examined. First, fibroblast-populated collagen lattice (FPCL) contraction was examined because it has been studied as a wound-healing model. In floating FPCL contraction assays, SS12 cells caused increased contraction, whereas AS10 cells caused reduced contraction. Because floating matrix contraction is thought to be mediated by the tractional force of the cells, cell behaviors related to tractional force were examined. In collagen matrix, SS12 cells elongated faster and to a greater extent and contained longer stress fibers than control cells, whereas AS10 cells were slower to elongate than control cells. SS12 cells attached to the dishes more efficiently than the control, whereas AS10 cells attached less efficiently. Migration of SS12 cells on collagen-coated dishes was also enhanced, although AS10 cells did not differ from the control cells. In summary, HSP27 regulates fibroblast adhesion, elongation, and migration and the contraction of the floating matrix in a manner dependent on the level of its expression.

Glutathione enhances fibroblast collagen contraction and protects keratinocytes from apoptosis in hyperglycaemic culture.

Background  Cutaneous wound healing is relatively slow in patients with diabetes.

Role of glutathione redox dysfunction in diabetic wounds

We propose that diabetic foot ulcers and diabetic mouse wounds have insufficient glutathione to maintain correct cellular redox potential. Therefore, tissue samples from the wound edge of diabetic foot ulcers, diabetic mice wounds and nondiabetic mice wounds were obtained. Levels of glutathione, cysteine, and mixed protein disulfide were determined and topical application of esterified glutathione in carboxymethylcellulose or carboxymethylcellulose alone was applied to the mice wounds. Diabetic foot ulcer mean glutathione levels were 150.6 pmol/mg in the controls and 53.4 pmol/mg at the wound edge (p < 0.05), while mean cysteine levels were 22.3 pmol/mg in the control and 10.5 pmol/mg at the wound edge (p < 0.05). The mixed protein disulfide levels were elevated in the wounds (14.6 pmol/mg), but not in the control (6.9 pmol/mg) (p < 0.05). The glutathione levels were lower in the diabetic mouse wounds (155 pmol/mg) than the nondiabetic mouse wounds (205 pmol/mg) (p=0.04). The diabetic mouse treated with carboxymethylcellulose alone healed slower (19.5 ± 2.2 days) than the nondiabetic mouse DM (11.5 ± 0.5 days) (p < 0.001). The diabetic mouse that received topical glutathione healed significantly faster (12.5 ± 0.8 days) than the carboxymethylcellulose‐treated mice (19.5 ± 2.2 days) (p < 0.001). Glutathione levels in the diabetic mouse (26.0 pmol/mg) were lower than in the nondiabetic mouse (311.7 pmol/mg) (p < 0.05) after glutathione treatment. In the glutathione‐treated diabetic mouse, the oxidized glutathione was higher (26.7%) than in the nondiabetic mouse (9.9%) (p=0.05). These data suggest that cellular redox dysfunction and lower glutathione levels are present in diabetic foot ulcers and diabetic mouse wounds.

Successful implantation of physiologically functional bioengineered mouse internal anal sphincter

We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered rings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence.

Surgical implantation of a bioengineered internal anal sphincter

PURPOSE Fecal incontinence is a common disorder that can have devastating social and psychologic consequences. However, there are no long-term ideal solutions for such patients. Although loss of continence is multifactorial, the integrity of the internal anal sphincter (IAS) has particular significance. We previously described the development of 3-dimensional bioengineered constructs using isolated smooth muscle tissue from donor C57BL/6 IAS. We hypothesized that the bioengineered ring constructs would retain cellular viability and promote neovascularization upon implantation into a recipient mouse. METHODS Internal anal sphincter ring constructs were surgically implanted into the subcutaneous tissue of syngeneic C57BL/6 mice and treated with either fibroblastic growth factor 2 (0.26 microg daily) or saline controls using a microosmotic pump. Internal anal sphincter constructs were harvested after 25 days (range, 23-26 days) and assessed morphologically and for tissue viability. RESULT Gross morphology showed that there was no rejection. Rings showed muscle attachment to the back of the mouse with no sign of inflammation. Fibroblastic growth factor 2 infusion resulted in a significantly improved histologic score and muscle viability compared with the control group. CONCLUSIONS Three-dimensional bioengineered IAS rings can be successfully implanted into the subcutaneous tissue of recipient mice. The addition of fibroblastic growth factor 2 led to improved muscle viability, vascularity, and survival. This approach may become a feasible option for patients with fecal incontinence.

Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle

Small heat shock proteins HSP27 and HSP20 have been implicated in regulation of contraction and relaxation in smooth muscle. Activation of PKC-α promotes contraction by phosphorylation of HSP27 whereas activation of PKA promotes relaxation by phosphorylation of HSP20 in colonic smooth muscle cells (CSMC). We propose that the balance between the phosphorylation states of HSP27 and HSP20 represents a molecular signaling switch for contraction and relaxation. This molecular signaling switch acts downstream on a molecular mechanical switch [tropomyosin (TM)] regulating thin-filament dynamics. We have examined the role of phosphorylation state(s) of HSP20 on HSP27-mediated thin-filament regulation in CSMC. CSMC were transfected with different HSP20 phosphomutants. These transfections had no effect on the integrity of actin cytoskeleton. Cells transfected with 16D-HSP20 (phosphomimic) exhibited inhibition of acetylcholine (ACh)-induced contraction whereas cells transfected with 16A-HSP20 (nonphosphorylatable) had no effect on ACh-induced contraction. CSMC transfected with 16D-HSP20 cDNA showed significant decreases in 1) phosphorylation of HSP27 (ser78); 2) phosphorylation of PKC-α (ser657); 3) phosphorylation of TM and CaD (ser789); 4) ACh-induced phosphorylation of myosin light chain; 5) ACh-induced association of TM with HSP27; and 6) ACh-induced dissociation of TM from caldesmon (CaD). We thus propose the crucial physiological relevance of molecular signaling switch (phosphorylation state of HSP27 and HSP20), which dictates 1) the phosphorylation states of TM and CaD and 2) their dissociations from each other.

VIP induces PKA-mediated rapid and sustained phosphorylation of HSP20

The small molecular weight heat shock protein HSP20 has been proposed to regulate smooth muscle relaxation in a manner dependent on its phosphorylated state. We present the first evidence of HSP20 phosphorylation in response to a naturally occurring neurotransmitter. HSP20 was rapidly phosphorylated in colonic circular smooth muscle cells exposed to the physiologically relevant relaxant neuropeptide, Vasoactive Intestinal Peptide (VIP). HSP20 phosphorylation was significantly and substantially increased by 30s following VIP treatment and remained elevated for 30 min. VIP-induced HSP20 phosphorylation was dose dependent. Both basal and VIP-induced HSP20 phosphorylations were solely mediated by Protein Kinase A. Maximal phosphorylation of HSP20 was induced by the same VIP concentration range which induces maximal relaxation. Increased phosphorylation of HSP20 occurred in both cytosolic and particulate cell fractions. Our findings represent evidence for neurogenic modulation of the cyclic molecular regulation of relaxation required for peristalsis via a VIP-PKA-HSP20 pathway.