Dehydrated human amniotic membrane regulates tenocyte expression and angiogenesis in vitro: Implications for a therapeutic treatment of tendinopathy.

Tendon injuries are among the most common ailments of the musculoskeletal system. Prolonged inflammation and persistent vasculature are common complications associated with poor healing. Damaged tendon, replaced with scar tissue, never completely regains the native structural or biomechanical properties. This study evaluated the effects of micronized dehydrated human amnion/chorion membrane (µdHACM) on the inflammatory environment and hypervascularity associated with tendinopathy. Stimulation of human tenocytes with interleukin-1 beta (IL1ß) induced the expression of inflammatory and catabolic markers, resulting in secretion of active MMPs and type 3 collagen that is associated with a degenerative phenotype. Treatment with µdHACM diminished the effects of IL1ß, reducing the expression of inflammatory genes, proteases, and extracellular matrix components, and decreasing the presence of active MMP and type 3 collagen. Additionally, a co-culture model was developed to evaluate the effects of µdHACM on angiogenesis associated with tendinopathy. Micronized dHACM differentially regulated angiogenesis depending upon the cellular environment in which it was placed. This phenomenon can be explained in part through the detection of both angiogenic protagonists and antagonists in µdHACM. Observations from this study identify a mechanism by which µdHACM regulates inflammatory processes and angiogenesis in vitro, two key pathways implicated in tendinopathic injuries.

Dehydrated Human Amniotic Membrane Inhibits Myofibroblast Contraction through the Regulation of the TGFß‒SMAD Pathway In Vitro.

Excessive fibrosis affects more than 100 million patients yearly, leading to the accumulation of extracellular matrix that compromises tissue architecture and impedes its function. Intrinsic properties of the amniotic membrane have alluded to its potential to inhibit excessive fibrosis; therefore, this study aimed to investigate the effects of dehydrated human amnion/chorion membrane (dHACM) on dermal fibroblasts and their role in fibrotic pathways. Human dermal fibroblasts were stimulated with TGFß1, triggering myofibroblast-like characteristics in vitro. Subsequent addition of dHACM in the continued presence of TGFß1 inhibited downstream signaling, leading to a reduction in the expression of known fibrotic and extracellular matrix genes. In addition, dHACM decreased alpha-smooth muscle actin, a stress filament responsible for contractile activity in scarring. The functional outcome of these effects was observed in an ex vivo model for cellular contraction. Hyperactivation of TGFß signaling increased the contractile capacity of myofibroblasts embedded within a collagen substrate. Simultaneous addition of dHACM treatment prevented the marked contraction, which is likely a direct result of the inhibition of TGFß signaling mentioned earlier. These observations may support the use of dHACM in the regulation of fibroblast activity as it relates to tissue fibrosis.

Dehydrated human amniotic membrane modulates canonical Wnt signaling in multiple cell types in vitro.

Canonical Wnt signaling is a major pathway known to regulate diverse physiological processes in multicellular organisms. Signaling is tightly regulated by feedback mechanisms; however, persistent dysregulation of this pathway is implicated in the progression of multiple disease states. In this study, proteomic analysis identified endogenous Wnt antagonists in micronized dehydrated human amnion/chorion membrane (µdHACM); thereby, prompting a study to further characterize the intrinsic properties of µdHACM as it relates to Wnt activity, in vitro. A TCF/LEF reporter cell line demonstrated the general ability of µdHACM to inhibit ß-catenin induced transcription activity. Furthermore, in vitro systems, modeling elevated Wnt signaling, were developed in relevant cell types including tenocytes, synoviocytes, and human dermal fibroblasts (HDFs). Stimulation of these cells with Wnt3A resulted in translocation of ß-catenin to the nucleus and increased expression of Wnt related genes. The subsequent addition of µdHACM, in the continued presence of Wnt-stimulus, mitigated the downstream effects of Wnt3A in tenocytes, synoviocytes, and HDFs. Nuclear localization of ß-catenin was abated with corresponding reduction of Wnt related gene expression. These data demonstrate the in vitro regulation of canonical Wnt signaling as an inherent property of µdHACM and a novel mechanism of action.

Differences in the timing and magnitude of Pkd1 gene deletion determine the severity of polycystic kidney disease in an orthologous mouse model of ADPKD.

Development of a disease-modifying therapy to treat autosomal dominant polycystic kidney disease (ADPKD) requires well-characterized preclinical models that accurately reflect the pathology and biochemical changes associated with the disease. Using a Pkd1 conditional knockout mouse, we demonstrate that subtly altering the timing and extent of Pkd1 deletion can have a significant impact on the origin and severity of kidney cyst formation. Pkd1 deletion on postnatal day 1 or 2 results in cysts arising from both the cortical and medullary regions, whereas deletion on postnatal days 3-8 results in primarily medullary cyst formation. Altering the extent of Pkd1 deletion by modulating the tamoxifen dose produces dose-dependent changes in the severity, but not origin, of cystogenesis. Limited Pkd1 deletion produces progressive kidney cystogenesis, accompanied by interstitial fibrosis and loss of kidney function. Cyst growth occurs in two phases: an early, rapid growth phase, followed by a later, slow growth period. Analysis of biochemical pathway changes in cystic kidneys reveals dysregulation of the cell cycle, increased proliferation and apoptosis, activation of Mek-Erk, Akt-mTOR, and Wnt-ß-catenin signaling pathways, and altered glycosphingolipid metabolism that resemble the biochemical changes occurring in human ADPKD kidneys. These pathways are normally active in neonatal mouse kidneys until repressed around 3 weeks of age; however, they remain active following Pkd1 deletion. Together, this work describes the key parameters to accurately model the pathological and biochemical changes associated with ADPKD in a conditional mouse model.

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.