The Pro-Fibrotic Response to Lens Injury is Signaled in a PI3K Isoform-Specific Manner

The signaling inputs that function to integrate biochemical and mechanical cues from the extracellular environment to alter the wound-repair outcome to a fibrotic response remain poorly understood. Here, using a clinically relevant post-cataract surgery wound healing/fibrosis model, we investigated the role of Phosphoinositide-3-kinase (PI3K) class I isoforms as potential signaling integrators to promote the proliferation, emergence and persistence of collagen I-producing alpha smooth muscle actin (αSMA+) myofibroblasts that cause organ fibrosis. Using PI3K isoform specific small molecule inhibitors, our studies revealed a requisite role for PI3K p110α in signaling the CD44+ mesenchymal leader cell population that we previously identified as resident immune cells to produce and organize a fibronectin-EDA rich provisional matrix and transition to collagen I-producing αSMA+ myofibroblasts. While the PI3K effector Akt was alone insufficient to regulate myofibroblast differentiation, our studies revealed a role for Rac, another potential PI3K effector, in this process. Our studies further uncovered a critical role for PI3K p110α in signaling the proliferation of CD44+ leader cells, which is important to the emergence and expansion of myofibroblasts. Thus, these studies identify activation of PI3K p110α as a critical signaling input following wounding to the development and progression of fibrotic disease

PI3K Isoform-Specific Regulation of Leader and Follower Cell Function for Collective Migration and Proliferation in Response to Injury

To ensure proper wound healing it is important to elucidate the signaling cues that coordinate leader and follower cell behavior to promote collective migration and proliferation for wound healing in response to injury. Using an ex vivo post-cataract surgery wound healing model we investigated the role of class I phosphatidylinositol-3-kinase (PI3K) isoforms in this process. Our findings revealed a specific role for p110α signaling independent of Akt for promoting the collective migration and proliferation of the epithelium for wound closure. In addition, we found an important role for p110α signaling in orchestrating proper polarized cytoskeletal organization within both leader and wounded epithelial follower cells to coordinate their function for wound healing. p110α was necessary to signal the formation and persistence of vimentin rich-lamellipodia extensions by leader cells and the reorganization of actomyosin into stress fibers along the basal domains of the wounded lens epithelial follower cells for movement. Together, our study reveals a critical role for p110α in the collective migration of an epithelium in response to wounding

The Pro-fibrotic Response of Mesenchymal Leader Cells to Lens Wounding Involves Hyaluronic Acid, Its Receptor RHAMM, and Vimentin

Hyaluronic Acid/Hyaluronan (HA) is a major component of the provisional matrix deposited by cells post-wounding with roles both in regulating cell migration to repair a wound and in promoting a fibrotic outcome to wounding. Both are mediated through its receptors CD44 and RHAMM. We now showed that HA is present in the provisional matrix assembled on the substrate surface in a lens post-cataract surgery explant wound model in which mesenchymal leader cells populate the wound edges to direct migration of the lens epithelium across the adjacent culture substrate onto which this matrix is assembled. Inhibiting HA expression with 4-MU blocked assembly of FN-EDA and collagen I by the wound-responsive mesenchymal leader cells and their migration. These cells express both the HA receptors CD44 and RHAMM. CD44 co-localized with HA at their cell-cell interfaces. RHAMM was predominant in the lamellipodial protrusions extended by the mesenchymal cells at the leading edge, and along HA fibrils organized on the substrate surface. Within a few days post-lens wounding the leader cells are induced to transition to αSMA+ myofibroblasts. Since HA/RHAMM is implicated in both cell migration and inducing fibrosis we examined the impact of blocking HA synthesis on myofibroblast emergence and discovered that it was dependent on HA. While RHAMM has not been previously linked to the intermediate filament protein vimentin, our studies with these explant cultures have shown that vimentin in the cells\u27 lamellipodial protrusions regulate their transition to myofibroblast. PLA studies now revealed that RHAMM was complexed with both HA and vimentin in the lamellipodial protrusions of leader cells, implicating this HA/RHAMM/vimentin complex in the regulation of leader cell function post-wounding, both in promoting cell migration and in the transition of these cells to myofibroblasts. These results increase our understanding of how the post-wounding matrix environment interacts with receptor/cytoskeletal complexes to determine whether injury outcomes are regenerative or fibrotic

Fibrosis-the Tale of H3K27 Histone Methyltransferases and Demethylases

Fibrosis, or excessive scarring, is characterized by the emergence of alpha-smooth muscle actin (αSMA)-expressing myofibroblasts and the excessive accumulation of fibrotic extracellular matrix (ECM). Currently, there is a lack of effective treatment options for fibrosis, highlighting an unmet need to identify new therapeutic targets. The acquisition of a fibrotic phenotype is associated with changes in chromatin structure, a key determinant of gene transcription activation and repression. The major repressive histone mark, H3K27me3, has been linked to dynamic changes in gene expression in fibrosis through alterations in chromatin structure. H3K27-specific homologous histone methylase (HMT) enzymes, Enhancer of zeste 1 and 2 (EZH1, EZH2), which are the alternative subunits of the Polycomb Repressive Complex 2 (PRC2) and demethylase (KDM) enzymes, Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and Lysine demethylase 6B (KDM6B), are responsible for regulating methylation status of H3K27me3. In this review, we explore how these key enzymes regulate chromatin structure to alter gene expression in fibrosis, highlighting them as attractive targets for the treatment of fibrosis

Resident immune cells of the avascular lens: Mediators of the injury and fibrotic response of the lens.

Tissues typically harbor subpopulations of resident immune cells that function as rapid responders to injury and whose activation leads to induction of an adaptive immune response, playing important roles in repair and protection. Since the lens is an avascular tissue, it was presumed that it was absent of resident immune cells. Our studies now show that resident immune cells are a shared feature of the human, mouse, and chicken lens epithelium. These resident immune cells function as immediate responders to injury and rapidly populate the wound edge following mock cataract surgery to function as leader cells. Many of these resident immune cells also express MHCII providing them with antigen presenting ability to engage an adaptive immune response. We provide evidence that during development immune cells migrate on the ciliary zonules and localize among the equatorial epithelial cells of the lens adjacent to where the ciliary zonules associate with the lens capsule. These findings suggest that the vasculature-rich ciliary body is a source of lens resident immune cells. We identified a major role for these cells as rapid responders to wounding, quickly populating each wound were they can function as leaders of lens tissue repair. Our findings also show that lens resident immune cells are progenitors of myofibroblasts, which characteristically appear in response to lens cataract surgery injury, and therefore, are likely agents of lens pathologies to impair vision like fibrosis

The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's-like disease

Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin plays pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating organ size, cell proliferation and differentiation. We now show that Hippo signaling is required for SMG branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and α-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from NOD mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and CTGF, known downstream targets of TAZ. Our studies show that Hippo signaling plays a crucial role in SMG branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans

Application of Terahertz Radiation to the Detection of Corrosion under the Shuttle's Thermal Protection System

There is currently no method for detecting corrosion under Shuttle tiles except for the expensive process of tile removal and replacement; hence NASA is investigating new NDE methods for detecting hidden corrosion. Time domain terahertz radiation has been applied to corrosion detection under tiles in samples ranging from small lab samples to a Shuttle with positive results. Terahertz imaging methods have been able to detect corrosion at thicknesses of 5 mils or greater under 1" thick Shuttle tiles and 7-12 mils or greater under 2" thick Shuttle tiles

Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland.

The development of ductal structures during branching morphogenesis relies on signals that specify ductal progenitors to set up a pattern for the ductal network. Here, we identify cellular asymmetries defined by the F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of duct specification in the embryonic submandibular gland (SMG). Apical polarity protein aPKCζ is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and further define the path of duct specification. Moreover, the motor protein myosin IIB, a mediator of mechanical force transmission along actin filaments, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. These studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG

Changes in Nascent Chromatin Structure Regulate Activation of the Pro-fibrotic Transcriptome and Myofibroblast Emergence in Organ Fibrosis

Cell reprogramming to a myofibroblast responsible for the pathological accumulation of extracellular matrix is fundamental to the onset of fibrosis. Here, we explored how condensed chromatin structure marked by H3K72me3 becomes modified to allow for activation of repressed genes to drive emergence of myofibroblasts. In the early stages of myofibroblast precursor cell differentiation, we discovered that H3K27me3 demethylase enzymes UTX/KDM6B creates a delay in the accumulation of H3K27me3 on nascent DNA revealing a period of decondensed chromatin structure. This period of decondensed nascent chromatin structure allows for binding of pro-fibrotic transcription factor, Myocardin-related transcription factor A (MRTF-A) to nascent DNA. Inhibition of UTX/KDM6B enzymatic activity condenses chromatin structure, prevents MRTF-A binding, blocks activation of the pro-fibrotic transcriptome, and results in an inhibition of fibrosis in lens and lung fibrosis models. Our work reveals UTX/KDM6B as central coordinators of fibrosis, highlighting the potential to target its demethylase activity to prevent organ fibrosis

High Diet Quality Is Associated with a Lower Risk of Cardiovascular Disease and All-Cause Mortality in Older Men

Although diet quality is implicated in cardiovascular disease (CVD) risk, few studies have investigated the relation between diet quality and the risks of CVD and mortality in older adults. This study examined the prospective associations between dietary scores and risk of CVD and all-cause mortality in older British men. A total of 3328 men (aged 60–79 y) from the British Regional Heart Study, free from CVD at baseline, were followed up for 11.3 y for CVD and mortality. Baseline food-frequency questionnaire data were used to generate 2 dietary scores: the Healthy Diet Indicator (HDI), based on WHO dietary guidelines, and the Elderly Dietary Index (EDI), based on a Mediterranean-style dietary intake, with higher scores indicating greater compliance with dietary recommendations. Cox proportional hazards regression analyses assessed associations between quartiles of HDI and EDI and risk of all-cause mortality, CVD mortality, CVD events, and coronary heart disease (CHD) events. During follow-up, 933 deaths, 327 CVD deaths, 582 CVD events, and 307 CHD events occurred. Men in the highest compared with the lowest EDI quartile had significantly lower risks of all-cause mortality (HR:0.75; 95% CI: 0.60, 0.94; P-trend = 0.03), CVD mortality (HR: 0.63; 95% CI: 0.42, 0.94; P-trend = 0.03), and CHD events(HR: 0.66; 95% CI: 0.45,0.97; P-trend = 0.05) but not CVD events (HR: 0.79; 95% CI: 0.60, 1.05;P-trend = 0.16) after adjustment for sociodemographic, behavioral, and cardiovascular risk factors. The HDI was not significantly associated with any of the outcomes. The EDI appears to be more useful than the HDI for assessing diet quality in relation to CVD and morality risk in older men. Encouraging older adults to adhere to the guidelines inherent in the EDI criteria may have public health benefit