FAK and p130Cas modulate stiffness-mediated early transcription and cellular metabolism.

Cellular metabolism is influenced by the stiffness of the extracellular matrix. Focal adhesion kinase (FAK) and its binding partner, p130Cas, transmit biomechanical signals about substrate stiffness to the cell to regulate a variety of cellular responses, but their roles in early transcriptional and metabolic responses remain largely unexplored. We cultured mouse embryonic fibroblasts with or without siRNA-mediated FAK or p130Cas knockdown and assessed the early transcriptional responses of these cells to placement on soft and stiff substrates by RNA sequencing and bioinformatics analyses. Exposure to the stiff ECM altered the expression of genes important for metabolic and biosynthetic processes, and these responses were influenced by knockdown of FAK and p130Cas. Our findings reveal that FAK-p130Cas signaling mechanotransduces ECM stiffness to early transcriptional changes that alter cellular metabolism and biosynthesis.

Survivin as a mediator of stiffness-induced cell cycle progression and proliferation of vascular smooth muscle cells.

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers a phenotypic switch, hypermigration, and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation. However, the mechanism underlying this trigger remains unknown. Our analyses of whole-transcriptome microarray data from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology identified 623 genes that were significantly and differentially expressed (360 upregulated and 263 downregulated) relative to expression in VSMCs cultured on soft hydrogels. Functional enrichment and gene network analyses revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Importantly, we found that survivin, an inhibitor of apoptosis protein, mediates stiffness-dependent cell cycle progression and proliferation as determined by gene network and pathway analyses, RT-qPCR, immunoblotting, and cell proliferation assays. Furthermore, we found that inhibition of cell cycle progression did not reduce survivin expression, suggesting that survivin functions as an upstream regulator of cell cycle progression and proliferation in response to ECM stiffness. Mechanistically, we found that the stiffness signal is mechanotransduced via the FAK-E2F1 signaling axis to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Overall, our findings indicate that survivin is necessary for VSMC cycling and proliferation and plays a role in regulating stiffness-responsive phenotypes.

Survivin regulates intracellular stiffness and extracellular matrix production in vascular smooth muscle cells.

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs significantly decreased the stiffness-mediated expression of ECM components related to arterial stiffening, such as collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these ECM proteins was rescued by ectopic expression of survivin in human VSMCs cultured on soft hydrogels. Interestingly, atomic force microscopy analysis showed that suppressed or ectopic expression of survivin decreases or increases intracellular stiffness, respectively. Furthermore, we observed that inhibiting Rac and Rho reduces survivin expression, elucidating a mechanical pathway connecting intracellular tension, mediated by Rac and Rho, to survivin induction. Finally, we found that survivin inhibition decreases FAK phosphorylation, indicating that survivin-dependent intracellular tension feeds back to maintain signaling through FAK. These findings suggest a novel mechanism by which survivin potentially modulates arterial stiffness.

Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3.

The Rho family GTPases Rac and Rho play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well-described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac was dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified activating transcription factor 3 (ATF3) as a major target of stiffness- and Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1.

Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation.

Cell cycle control is a key aspect of numerous physiological and pathological processes. The contribution of biophysical cues, such as stiffness or elasticity of the underlying extracellular matrix (ECM), is critically important in regulating cell cycle progression and proliferation. Indeed, increased ECM stiffness causes aberrant cell cycle progression and proliferation. However, the molecular mechanisms that control these stiffness-mediated cellular responses remain unclear. Here, we address this gap and show good evidence that lamellipodin (symbol RAPH1), previously known as a critical regulator of cell migration, stimulates ECM stiffness-mediated cyclin expression and intracellular stiffening in mouse embryonic fibroblasts. We observed that increased ECM stiffness upregulates lamellipodin expression. This is mediated by an integrin-dependent FAK-Cas-Rac signaling module and supports stiffness-mediated lamellipodin induction. Mechanistically, we find that lamellipodin overexpression increased, and lamellipodin knockdown reduced, stiffness-induced cell cyclin expression and cell proliferation, and intracellular stiffness. Overall, these results suggest that lamellipodin levels may be critical for regulating cell proliferation. This article has an associated First Person interview with the first author of the paper.

Emerging machine learning approaches to phenotyping cellular motility and morphodynamics.

Cells respond heterogeneously to molecular and environmental perturbations. Phenotypic heterogeneity, wherein multiple phenotypes coexist in the same conditions, presents challenges when interpreting the observed heterogeneity. Advances in live cell microscopy allow researchers to acquire an unprecedented amount of live cell image data at high spatiotemporal resolutions. Phenotyping cellular dynamics, however, is a nontrivial task and requires machine learning (ML) approaches to discern phenotypic heterogeneity from live cell images. In recent years, ML has proven instrumental in biomedical research, allowing scientists to implement sophisticated computation in which computers learn and effectively perform specific analyses with minimal human instruction or intervention. In this review, we discuss how ML has been recently employed in the study of cell motility and morphodynamics to identify phenotypes from computer vision analysis. We focus on new approaches to extract and learn meaningful spatiotemporal features from complex live cell images for cellular and subcellular phenotyping.

Phosphoinositide Signaling and Mechanotransduction in Cardiovascular Biology and Disease.

Phosphoinositides, which are membrane-bound phospholipids, are critical signaling molecules located at the interface between the extracellular matrix, cell membrane, and cytoskeleton. Phosphoinositides are essential regulators of many biological and cellular processes, including but not limited to cell migration, proliferation, survival, and differentiation, as well as cytoskeletal rearrangements and actin dynamics. Over the years, a multitude of studies have uniquely implicated phosphoinositide signaling as being crucial in cardiovascular biology and a dominant force in the development of cardiovascular disease and its progression. Independently, the cellular transduction of mechanical forces or mechanotransduction in cardiovascular cells is widely accepted to be critical to their homeostasis and can drive aberrant cellular phenotypes and resultant cardiovascular disease. Given the versatility and diversity of phosphoinositide signaling in the cardiovascular system and the dominant regulation of cardiovascular cell functions by mechanotransduction, the molecular mechanistic overlap and extent to which these two major signaling modalities converge in cardiovascular cells remain unclear. In this review, we discuss and synthesize recent findings that rightfully connect phosphoinositide signaling to cellular mechanotransduction in the context of cardiovascular biology and disease, and we specifically focus on phosphatidylinositol-4,5-phosphate, phosphatidylinositol-4-phosphate 5-kinase, phosphatidylinositol-3,4,5-phosphate, and phosphatidylinositol 3-kinase. Throughout the review, we discuss how specific phosphoinositide subspecies have been shown to mediate biomechanically sensitive cytoskeletal remodeling in cardiovascular cells. Additionally, we discuss the direct interaction of phosphoinositides with mechanically sensitive membrane-bound ion channels in response to mechanical stimuli. Furthermore, we explore the role of phosphoinositide subspecies in association with critical downstream effectors of mechanical signaling in cardiovascular biology and disease.

Radiographic measurement of intestinal length among children with short bowel syndrome: Retrospective determination remains problematic.

PURPOSE: Small bowel length is the most reliable predictor of enteral independence in pediatric short bowel syndrome. Retrospectively measured bowel lengths on upper GI with small bowel follow-through (UGI/SBFT) were compared to operative measurements. METHODS: A pediatric radiologist and surgical trainees blinded to operative measurements retrospectively analyzed UGI/SBFT studies using the digital radiography curved measurement tool. Children with SBS and severe intestinal failure (parenteral nutrition >90days) at a multidisciplinary intestinal failure program 2002-2015 were included. Data were expressed as median (Q1, Q3). RESULTS: Thirty-six children aged 0.8 (0.4, 3.7) years were analyzed. Fifty-six percent had intestinal malrotation, and 58% had prior serial transverse enteroplasty. Studies were conducted within 10 (7, 20) days of surgery. Intraoperative bowel length was 90cm (45, 142), while UGI/SBFT measurement by radiologist was 45cm (28, 63), with a mean difference of 47cm (SD 58cm, p<0.001) and a mean percent error of 50%. Radiographic assessment underestimated intestinal length in 83% of patients. CONCLUSION: Bowel length measured retrospectively from upper GI with small bowel follow-through studies usually underestimated intraoperative bowel length. The limits of agreement were too wide for this technique to be clinically useful. Operative measurement remains necessary to assess intestinal length and rehabilitation potential. TYPE OF STUDY: Study of Diagnostic Test. LEVEL OF EVIDENCE: Level III.

Comparisons of human amniotic mesenchymal stem cell viability in FDA-approved collagen-based scaffolds: Implications for engineered diaphragmatic replacement.

BACKGROUND/PURPOSE: We sought to examine amniotic fluid mesenchymal stem cell (afMSC) viability within two FDA-approved collagen-based scaffolds, as a prerequisite to clinical translation of afMSC-based engineered diaphragmatic repair. METHODS: Human afMSCs were seeded in a human-derived collagen hydrogel and in a bovine-derived collagen sheet at 3 matching densities. Cell viability was analyzed at 1, 3, and 5days using an ATP-based 3D bioluminescence assay. Statistical comparisons were by ANOVA (P<0.05). RESULTS: There was a highly significant 3-way interaction between scaffold type, seeding density, and time in 3D culture as determinants of cell viability, clearly favoring the human hydrogel (P<0.001). In both scaffolds, cell viability was highest at the highest seeding density of 150,000 cells/mL. Time in 3D culture impacted cell viability at the optimal seeding density in the human hydrogel, with the highest levels on days 1 (P<0.001) and 5 (P=0.05) with no significant effect in the bovine sheet (P=0.39-0.96). CONCLUSIONS: Among clinically-approved cell delivery vehicles, mesenchymal stem cell viability is significantly enhanced in a collagen hydrogel when compared with a collagen sheet. Cell viability can be further optimized by seeding density and time in 3D culture. These data further support the regulatory viability of clinical trials of engineered diaphragmatic repair. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Donor mesenchymal stem cells home to maternal wounds after transamniotic stem cell therapy (TRASCET) in a rodent model.

PURPOSE: Transamniotic stem cell therapy (TRASCET) with amniotic fluid-derived MSCs (afMSCs) has emerged experimentally as a practical treatment strategy for congenital anomalies. In this study, we sought to determine whether afMSCs migrate to the mother following TRASCET. METHODS: Pregnant rat dams were divided into three groups. Two groups received volume-matched injections into all amniotic cavities of either a suspension of afMSCs labeled with a luciferase reporter gene or the luciferase protein alone. In a third group, a suspension of labeled cells was aliquoted onto the serosal surface of the uterus. Maternal samples from the laparotomy scar (fascia and skin separately), bone marrow, and peripheral blood were procured, along with placenta and umbilical cord. Specimens were screened for luminescence via microplate luminometry. RESULTS: Luminescence was detected in 60% (9/15) of the fascial scars from the group receiving intraamniotic injection of afMSCs, but in none of the other groups (P<0.001). There was a direct correlation between the presence of donor cells in the placenta and their presence in maternal fascia (Wald test=10.2; P=0.001). CONCLUSIONS: Amniotic mesenchymal stem cells migrate to maternal sites of injury after intraamniotic injection. Maternal homing of donor cells must be considered in the setting of transamniotic stem cell therapy. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Transamniotic stem cell therapy (TRASCET) in a leporine model of gastroschisis.

BACKGROUND/PURPOSE: Transamniotic stem cell therapy (TRASCET) with amniotic fluid mesenchymal stem cells (afMSCs) has been shown to mitigate bowel damage in a rodent model of gastroschisis. As a prerequisite to clinical translation, we sought to study TRASCET in a larger animal model. METHODS: New Zealand rabbit fetuses (n=64) with surgically created gastroschisis were divided into three groups. One group (untreated) had no further manipulations. Two groups received volume-matched intraamniotic injections of either saline or a suspension of afMSCs. Nonmanipulated fetuses served as controls. Histomorphologic measurements of intestinal damage, along with biochemical profiling of inflammation markers, were performed at term. Statistical comparisons were by Fisher's exact test, ANOVA and the Wald test (P<0.05). RESULTS: Overall survival was 62.5%. Segmental and total intestinal wall thicknesses were significantly decreased in the afMSC group compared with the untreated and saline groups (all P<0.001), with no significant differences between untreated and saline groups (P=0.24 to 1.00, depending on layer). Muscularis and serosal layers were significantly thicker in the afMSC group than in normal controls (P=0.045 and P<0.001, respectively). CONCLUSIONS: Concentrated intraamniotic injection of afMSC lessens, yet does not prevent, intestinal damage in a leporine model of gastroschisis. TRASCET may become a valuable strategy in the management of gastroschisis. LEVEL OF EVIDENCE: N/A - animal/experimental studies.

A comparison between placental and amniotic mesenchymal stem cells for transamniotic stem cell therapy (TRASCET) in experimental spina bifida.

PURPOSE: We compared placental-derived and amniotic fluid-derived mesenchymal stem cells (pMSCs and afMSCs, respectively) in transamniotic stem cell therapy (TRASCET) for experimental spina bifida. METHODS: Pregnant dams (n=29) exposed to retinoic acid for the induction of fetal spina bifida were divided into four groups. Three groups received volume-matched intraamniotic injections of either saline (n=38 fetuses) or a suspension of 2×10(6) cells/mL of syngeneic, labeled afMSCs (n=73) or pMSCs (n=115) on gestational day 17 (term=21-22days). Untreated fetuses served as controls. Animals were killed before term. Statistical comparisons were by Fisher's exact test (p<0.05). RESULTS: Survival was similar across treatment groups (p=0.08). In fetuses with isolated spina bifida (n=100), there were higher percentages of defect coverage (either partial or complete) in both afMSC and pMSC groups compared with saline and untreated groups (p<0.001-0.03 in pairwise comparisons). There were no differences in coverage rates between afMSC and pMSC groups (p=0.94) or between saline and untreated groups (p=0.98). CONCLUSIONS: Both pMSC and afMSC can induce comparable rates of coverage of experimental spina bifida after concentrated intraamniotic injection in the rodent model. This broadens the options for timing and cell source for TRASCET as a potential alternative in the prenatal management of spina bifida.

Transamniotic stem cell therapy (TRASCET) mitigates bowel damage in a model of gastroschisis.

PURPOSE: We sought to determine whether intraamniotic delivery of concentrated amniotic-derived mesenchymal stem cells (afMSCs) could reduce damage to exposed bowel in experimental gastroschisis. METHODS: Rat fetuses (n=117) with surgically created gastroschisis were divided into three groups: untreated animals (n=62) and two groups receiving volume-matched intraamniotic injections of either saline (n=25) or 2 × 10(6) cells/mL of syngeneic, labeled afMSCs (n=30). Animals were killed before term, along with normal controls (NL). Blinded observers performed computerized measurements of total and segmental (serosa, muscularis, and mucosa) intestinal wall thicknesses. Statistical comparisons were by ANOVA (P<0.05). RESULTS: Among survivors with gastroschisis, there were statistically significant decreases in total bowel wall, serosal, muscular, and mucosal thicknesses in the afMSC group vs. the untreated group (P=0.001/0.035/0.001/0.005, respectively) and vs. the saline group (P=0.003/0.05/<0.001/0.026, respectively). There were no such significant differences between the untreated and saline groups. There were no differences between the afMSC group and NL, except for a significantly thicker muscular layer in the afMSC group (P=0.014). Labeled afMSCs were scarcely identified, suggesting a paracrine effect. CONCLUSIONS: Amniotic mesenchymal stem cells mitigate bowel damage in experimental gastroschisis after concentrated intraamniotic injection. Transamniotic stem cell therapy (TRASCET) may become a practical component of the treatment of gastroschisis.

Trans-amniotic stem cell therapy (TRASCET) minimizes Chiari-II malformation in experimental spina bifida.

PURPOSE: We sought to study the impact of trans-amniotic stem cell therapy (TRASCET) in the Chiari-II malformation in experimental spina bifida. METHODS: Sprague-Dawley fetuses (n=62) exposed to retinoic acid were divided into three groups at term (21-22 days gestation): untreated isolated spina bifida (n=21), isolated spina bifida treated with intra-amniotic injection of concentrated, syngeneic, labeled amniotic fluid mesenchymal stem cells (afMSCs) on gestational day 17 (n=28), and normal controls (n=13). Analyses included measurements of brainstem and cerebellar placement on high resolution MRI and histology. Statistical comparisons included ANOVA. RESULTS: In parallel to the expected induced coverage of the spina bifida in the afMSC-treated group (P<0.001), there were statistically significant differences in brainstem displacement across the groups (P<0.001), with the highest caudal displacement in the untreated group. Significant differences in cerebellar displacement were also noted, albeit less pronounced. Pairwise comparisons were statistically significant, with P=0.014 between treated and normal controls in caudal brainstem displacement and P<0.001 for all other comparisons. Labeled afMSCs were identified in 71% of treated fetuses. CONCLUSIONS: Induced coverage of spina bifida by TRASCET minimizes the Chiari-II malformation in the retinoic acid rodent model, further suggesting it as a practical alternative for the prenatal management of spina bifida.

Extraluminal distraction enterogenesis using shape-memory polymer.

PURPOSE: Although a few techniques for lengthening intestine by mechanical stretch have been described, they are relatively complex, and the majority involve placement of an intraluminal device. Ideally, techniques applicable to humans would be easy to perform and extraluminal to avoid the potential for mucosal injury. This study of distraction enterogenesis used an extraluminal, radially self-expanding shape-memory polymer cylinder and a simple operative approach to both elongate intestine and grow new tissue. METHODS: Young Sprague Dawley rats (250-350 g) underwent Roux-en-Y isolation of a small intestinal limb and were divided in three groups: no further manipulation (Control 1, C1); placement of a nonexpanding device (Control 2, C2); or placement of a radially expanding device by the limb (Experimental, Exp). For C2 and Exp animals, the blind end of the limb was wrapped around the radially expanding cylindrical device with the limb-end sutured back to the limb-side. Bowel length was measured at operation and at necropsy (14 days) both in-situ and ex-vivo under standard tension (6g weight). Change in length is shown as mean ± standard deviation. A blinded gastrointestinal pathologist reviewed histology and recorded multiple measures of intestinal adaptation. The DNA to protein ratio was quantified as a surrogate for cellular proliferation. Changes in length, histologic measures, and DNA:protein were compared using analysis of variance, with significance set at P<0.05. RESULTS: The length of the Roux limb in situ increased significantly in Exp animals (n=8, 29.0 ± 5.8mm) compared with C1 animals (n=5, -11.2 ± 9.0mm, P<0.01). The length of the Roux limb ex vivo under standard tension increased in the Exp group (25.8 ± 4.2mm) compared with the C2 group (n=6, -4.3 ± 6.0, P<0.01). There were no differences in histologic measures of bowel adaptation between the groups, namely villous height and width, crypt depth, crypt density, and crypt fission rate (all P ≥ 0.08). Muscularis mucosal thickness was also not different (P=0.25). There was no difference in DNA:protein between groups (P=0.47). CONCLUSION: An extraluminally placed, radially expanding shape-memory polymer cylinder successfully lengthened intestine, without damaging mucosa. Lack of difference in muscularis thickness and a constant DNA:protein ratio suggests that this process may be related to actual growth rather than mere stretch. This study demonstrated a simple approach that warrants further study aiming at potential clinical applicability.

Limb reconstruction with decellularized, non-demineralized bone in a young leporine model.

Limb salvage from a variety of pathological processes in children is often limited by the unavailability of optimal allograft bone, or an appropriate structural bone substitute. In this study, we sought to examine a practical alternative for pediatric limb repair, based on decellularized, non-demineralized bone grafts, and to determine whether controlled recellularization prior to implantation has any impact on outcome. Growing New Zealand rabbits (n = 12) with a complete, critical-size defect on the left tibiofibula were equally divided into two groups. One group received a decellularized, non-demineralized leporine tibiofibula graft. The other group received an equivalent graft seeded with mesenchymal stem cells labeled with green fluorescent protein (GFP), at a fixed density. Animals were euthanized at comparable time points 3-8 weeks post-implantation. Statistical analysis was by the Student t-test and Fisher's exact test (P < 0.05). There was no significant difference in the rate of non-union between the two groups, including on 3D micro-CT. Incorporated grafts achieved adequate axial bending rigidity, torsional rigidity, union yield and flexural strength, with no significant differences or unequal variances between the groups. Correspondingly, there were no significant differences in extracellular calcium levels, or alkaline phosphatase activity. Histology confirmed the presence of neobone in both groups, with GFP-positive cells in the recellularized grafts. It was shown that osseous grafts derived from decellularized, non-demineralized bone undergo adequate remodeling in vivo after the repair of critical-size limb defects in a growing leporine model, irrespective of subsequent recellularization. This methodology may become a practical alternative for pediatric limb reconstruction.

Partial or complete coverage of experimental spina bifida by simple intra-amniotic injection of concentrated amniotic mesenchymal stem cells.

PURPOSE: We sought to determine whether simple intra-amniotic delivery of concentrated amniotic mesenchymal stem cells (afMSCs) may elicit prenatal coverage of experimental spina bifida. METHODS: Time-dated pregnant Sprague-Dawley dams (n=24) exposed to retinoic acid for the induction of fetal neural tube defects were divided in three groups. Group I had no further manipulations. Groups II and III received volume-matched intra-amniotic injections of either saline (Group II) or a suspension of syngeneic afMSCs labeled with green fluorescent protein (Group III) in all fetuses (n=202) on gestational day 17 (term=21-22 days). Animals were killed before term. Statistical comparisons were by ANOVA (P<0.05). RESULTS: Of 165 fetuses viable at euthanasia, a spina bifida was present in 58% (96/165), with no significant differences in defect dimension across the groups (P=0.19). However, variable degrees of coverage of the defect by a rudimentary skin confirmed histologically were only present in Group III (P<0.001), in which donor afMSCs were documented, with no differences between Groups I and II (P=0.98). CONCLUSIONS: Amniotic mesenchymal stem cells can induce partial or complete coverage of experimental spina bifida after concentrated intra-amniotic injection. Trans-amniotic stem cell therapy (TRASCET) may become a practical option in the prenatal management of spina bifida.

Extraluminal helicoidal stretch (Helixtretch): a novel method of intestinal lengthening.

PURPOSE: We sought to test a novel, extraluminal method of intestinal lengthening that precludes violation of the intestinal wall. METHODS: Sprague-Dawley rats (n=45) with size-matched bowel segments isolated by Roux-en-Y reconstruction were divided into three groups. Group 1 (n=14) had no further manipulations. In Groups 2 (n=12) and 3 (n=19), the isolated segment was wrapped around a length-matched device in a helicoidal fashion. In Group 2, the device consisted of plain polyurethane tubing. In Group 3, it consisted of a gradually expanding hygroscopic hydrogel (12.5mm final diameter). Euthanasia was performed at 8-21 days. Statistical analysis was by two-way ANOVA (P<0.05). RESULTS: Overall survival was 87% (39/45). There was a statistically significant increase in bowel length in Group 3 compared to the other two groups (P<0.001). This increase correlated with the number of helicoidal coils (P=0.018), but not with post-operative time (P>0.50). There were no significant differences in total DNA/protein ratio across the groups (P=0.65). Histologically, there was an apparent increase in the goblet cell density in Group 3. CONCLUSIONS: Measured extraluminal helicoidal stretch (Helixtretch) is tolerated by the intestine. Helixtretch induces bowel lengthening in a rodent model. Further analysis of this novel, minimally invasive alternative for intestinal augmentation is warranted.

Dynamic alterations in Hippo signaling pathway and YAP activation during liver regeneration.

The Hippo signaling pathway has been implicated in mammalian organ size regulation and tumor suppression. Specifically, the Hippo pathway plays a critical role regulating the activity of transcriptional coactivator Yes-associated protein (YAP), which modulates a proliferative transcriptional program. Recent investigations have demonstrated that while this pathway is activated in quiescent livers, its inhibition leads to liver overgrowth and tumorigenesis. However, the role of the Hippo pathway during the natural process of liver regeneration remains unknown. Here we investigated alterations in the Hippo signaling pathway and YAP activation during liver regeneration using a 70% partial hepatectomy (PH) rat model. Our results indicate an increase in YAP activation by 1 day following PH as demonstrated by increased YAP nuclear localization and increased YAP target gene expression. Investigation of the Hippo pathway revealed a decrease in the activation of core kinases Mst1/2 by 1 day as well as Lats1/2 and its adapter protein Mob1 by 3 days following PH. Evaluation of liver-to-body weight ratios indicated that the liver reaches its near normal size by 7 days following PH, which correlated with a return to baseline YAP nuclear levels and target gene expression. Additionally, when liver size was restored, Mst1/2 kinase activation returned to levels observed in quiescent livers indicating reactivation of the Hippo signaling pathway. These findings illustrate the dynamic changes in the Hippo signaling pathway and YAP activation during liver regeneration, which stabilize when the liver-to-body weight ratio reaches homeostatic levels.