Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

Abstract

<jats:p>Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca<jats:sup>2+</jats:sup>, KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and Gö-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K<jats:sup>+</jats:sup>depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and Gö-6850 (each 10<jats:sup>−5</jats:sup>M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs′ SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS.</jats:p>

Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans

“…In certain experiments, we also examined the effects of the inhibitors given in different combinations as outlined in RESULTS. Isolation of SMCs and cell culture. Smooth muscle cells (SMCs) from the IAS and RSM were isolated as described previously (38,43,45). Briefly, the circular smooth muscle tissues were minced into ϳ1-mm cubes with sharp scissors and kept for incubation in oxygenated KPS (containing 0.1% collagenase type I and 0.01% soybean trypsin inhibitor) at 37°C.…”

“…The cells were then stained with secondary antibodies (FITC and Texas red-conjugated) and with 4=,6-diamidino-2-phenylindole (DAPI) for nucleic acid staining as described before (45). The slides were then air dried and coverslipped with ProLong Gold mounting medium (Invitrogen, Carlsbad, CA).…”

“…The tissue samples were rinsed with PBS and suspended in ice-cold homogenization buffer (10 mM Tris·HCl, pH 7.5, 5 mM MgCl 2, 2 mM EDTA, 250 mM sucrose, and 1 mM dithiothreitol, 1% Triton X-100) and homogenized by using tissue homogenizer (IKA ultra, Turrax, Wilmington, DE). The tissue extracts were centrifuged at 800 g for 10 min, and protein concentrations in the resultant supernatants were determined by use of a BCA Protein Assay Reagent Kit (Pierce, Rockford, IL) (45).…”

Ca2+/calmodulin/MLCK pathway initiates, and RhoA/ROCK maintains, the internal anal sphincter smooth muscle tone

“…Studies using selective molecular intervention by localized topical application of ROCKII-siRNA for transient silencing of ROCKII also demonstrated a significant decrease in the IAS tone (4). Further evidence implicating the RhoA/ROCK pathway as responsible for the basal tone has emerged from studies of bioengineered and reverse engineered IAS reconstructs using human IAS SMCs (34). These reconstructs were shown to have functional and molecular properties similar to the intact IAS and demonstrated that the basal tone is dependent on RhoA/ROCK.…”

Role of differentially expressed microRNA-139-5p in the regulation of phenotypic internal anal sphincter smooth muscle tone

“…There is an agreement as well that unique myogenic properties of the smooth muscle cells (SMCs) in the IAS are primarily responsible for the basal tone 6–13 . However, molecular mechanisms underlying the IAS tone are not well understood.…”