Vascular calcification is a strong predictor of cardiovascular mortality and lacks effective treatment. The transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like phenotypes is a key driver of calcification. This study identifies a regulatory role for Hyaluronan (HA) in VSMC osteogenic differentiation and arterial calcification. Human aortic VSMCs stimulated with high phosphate and/or pro-inflammatory cytokines (IL6 and TGF-β1) exhibited increased RUNX2, alkaline phosphatase and osteopontin expression, along with reduced contractility and elevated calcium deposition. This corresponded with reduced HA deposition and downregulation of HA synthase enzymes (HAS1, HAS2), Hyaluronidase enzymes (Hyal1), and HA binding proteins (CD44, TSG-6), whilst HAS3 and versican were upregulated. Comparable alterations in HA and protein expression were observed in an in vivo model of arterial calcification using vitamin K-deficient warfarin-fed mice. Pharmacological inhibition of HA synthesis, enzyme-mediated HA degradation and siRNA/plasmid modulation of HAS isoenzymes demonstrated a possible functional link between HA regulation and VSMC osteogenic differentiation. This study establishes HA and its associated binding proteins as key regulators of arterial calcification, highlighting a novel pathway for potential therapeutic intervention.

Non-healing chronic wounds, such as venous ulcers and pressure sores, represent significant causes of patient morbidity and financial burden to Healthcare Services worldwide. During normal healing, dermal fibroblasts (DFs) mediate numerous responses to promote wound closure. However, phenotypic changes induced within chronic wound environments lead to dysfunctional fibroblast functions, which facilitate non-healing. Although the processes underlying impaired proliferative and migratory responses in chronic wound fibroblasts (CWFs) are established, the mechanisms that mediate impaired CWF-myofibroblast differentiation remain poorly understood. Fibroblast-myofibroblast differentiation is induced by transforming growth factor-β1 (TGF-β1) and downstream classical Smad2/3 and non-classical epidermal growth factor receptor (EGFR)/ERK1/2 signaling, initiated through hyaluronan (HA) receptor (CD44) binding to EGFR and dependent on elevated HA synthesis and its pericellular accumulation. Here, we demonstrate that these signaling pathways are dysregulated in venous ulcer- and pressure sore-derived CWFs, compared to DFs. CWFs exhibit increased susceptibilities to cellular senescence and impaired myofibroblast differentiation, accompanied by defective lysosomal/endosomal activities and dysfunctional activation of the HA/CD44/EGFR pathway. Irrespective of wound source, CWFs exhibited increased HAS1 versus HAS2 expression, altered HAS1 and HAS2 intracellular localization, and deregulated hyaladherin (CD44, TSG-6, and IαI heavy chain motifs, HC3, HC4 and HC5) induction, following TGF-β1 stimulation. These events attenuated HA pericellular coat formation and CD44/EGFR co-localization within membrane lipid rafts, essential for myofibroblast development. Our findings suggest that aberrant HAS1 and HAS2 expression and distributions cause reduced pericellular hyaluronan deposition, leading to attenuated CD44/EGFR co-localization and dysfunctional CWF-myofibroblast differentiation, which contributes to the impaired closure and healing of chronic wounds.

Epoxytiglianes are a novel class of diterpene esters. The prototype epoxytigliane, EBC-46 (tigilanol tiglate), is a potent anti-cancer agent in clinical development for local treatment of a range of human and animal tumors. EBC-46 also consistently promotes wound re-epithelialization at the treatment sites, mediated via activation of classical protein kinase C (PKC) isoforms. We have previously shown that epoxytiglianes stimulate proliferative and wound repopulation responses in immortalized human skin keratinocytes (HaCaTs) in vitro, abrogated by pan-PKC inhibitor, bisindolylmaleimide-1. In this study, we further investigate the specific PKC isoforms responsible for inducing such wound healing responses, following HaCaT treatment with 1.51 nM-15.1 mu M EBC46 or analogue, EBC-211. Classical PKC inhibition by GO6976 (1 mu M), significantly attenuated epoxytigliane induced, HaCaT proliferation and wound repopulation at all epoxytigliane concentrations. PKC-(3I/-(3II isoform inhibition by enzastaurin (1 mu M), significantly inhibited HaCaT proliferation and wound repopulation responses induced by both epoxytiglianes, especially at 1.51-151 nM. PKC-alpha inhibitor, Ro 31-8220 mesylate (10 nM), exerted lesser inhibitory effects on HaCaT responses. Epoxytigliane changes in key keratin (KRT17) and cell cycle (cyclin B1, CDKN1A) protein levels were partly attenuated by GO6976 and enzastaurin. GO6976 also inhibited increases in matrix metalloproteinase (MMP-1, MMP-7, MMP-10) activities. Phospho-PKC (p-PKC) studies confirmed that epoxytiglianes transiently activated classical PKC isoforms (p-PKC alpha, p-PKC-(3I/-(3II, p-PKC gamma) in a dose- and time-dependent manner. By identifying how epoxytiglianes stimulate classical PKCs to facilitate keratinocyte healing responses and re-epithelialization, these findings support further epoxytigliane development as topical therapeutics for clinical situations involving impaired re-epithelialization, such as non-healing wounds in skin.

ABSTRACT Background The stroma plays a key role during renal development and in regeneration after injury. However, following injury, the stroma expands driving progressive fibrosis. Hyaluronan (HA) is a glycosaminoglycan that is absent in healthy kidney stroma but highly expressed in disease. To understand strategies to modulate stromal HA towards therapeutic advantage, this study compares HA and HA Synthase (HAS) enzyme expression in kidney development, health, disease, and recovery. Methods Rats underwent ischaemia reperfusion injury (IRI) with/without ischaemic preconditioning (IPC) and kidneys histologically analysed. Kidneys from C57BL/6 embryos and HAS1/3 -/- mice were also analysed and parallel mechanistic cell studies performed using primary human fibroblasts. Results In health, stromal HA was absent from the renal cortex. HAS1 was expressed in some epithelial cells, whilst HAS2 was not expressed. Following IRI there was increased stromal HA in areas of chronic fibrosis, alongside increased HAS2 (but not HAS1) expression. In contrast, during development prominent stromal HA matrices were evident in areas of tubular generation, with strong HAS1 (not HAS2) expression. Following IPC+IRI, stromal HA and HAS2 were attenuated; whilst HAS1 + cells expanded but were distinct from α-SMA + myofibroblasts. Cell studies demonstrated that HAS1 + fibroblasts had a functionally distinct phenotype, with enhanced migration and FAP expression but attenuated α-SMA, EDA-FN and COL1A1 expression, whereas HAS2 + fibroblasts demonstrated a classic α-SMA + contractile myofibroblast phenotype with high EDA-FN and COL1A1. Conclusions HA is a key regulator of stromal fibroblast heterogeneity, with HAS1 and HAS2 defining phenotypically distinct populations that may influence divergent renal outcomes following injury. SIGNIFICANCE STATEMENT Hyaluronan (HA) is a matrix glycosaminoglycans that is absent in healthy kidney cortex but demonstrates increased expression in the renal stroma during progressive fibrosis. This study makes comparisons of HA accumulation, localisation, and HA Synthase (HAS) protein expression during kidney development, in health, following ischaemic kidney injury and during renal recovery. The study identifies that different HAS isoenzymes (HAS1 and HAS2) mediate distinct functional fibroblast phenotypes in vitro and are localised in distinct stromal localisations and cell sub-populations in vivo . The data provides interesting insights into HA dependent regulation of fibroblast stromal heterogeneity and identifies the novel finding that HAS1 defines cell populations that are associated with kidney recovery following ischaemic injury and are protective against progressive renal fibrosis.

OBJECTIVES:Acute Kidney Injury (AKI) and Chronic Kidney Disease (CKD) are increasingly recognised as one disease continuum, rather than distinct entities, and are associated with a huge burden to healthcare services. The leading cause of AKI worldwide is Ischaemia Reperfusion Injury (IRI), most commonly seen in clinical settings of sepsis-driven hypotension. Ischaemic Preconditioning (IPC) is a strategy aimed at reducing the deleterious effects of IRI. The objectives of this study were to demonstrate an efficacious in vivo model of Kidney IRI, and the protective influence of IPC in attenuating AKI and development of renal fibrosis.METHODS:A rat model of bilateral kidney IRI was used: Male Lewis rats (n=84) were assigned to IRI, sham or IPC. In IRI, renal pedicles were clamped for 45 minutes. IPC groups underwent pulsatile IPC prior to IRI. Kidneys were retrieved at 24 hours, 48 hours, 7 days, 14 days and 28 days, and assessed histologically.RESULTS:IRI led to marked AKI (24-48 h) and renal fibrosis development by 28 days. IPC attenuated this damage, with 66% less fibrosis. Interestingly, at 14-days, the histological appearance of both IRI and IPC kidneys was rather similar, potentially representing an important transitional point at which kidneys commit to either fibrosis or recovery. This may provide a suitable inflexion point for introduction of novel anti-fibrotic therapies.CONCLUSIONS:In conclusion, we have characterised a model of kidney injury from acute to chronic phases, allowing detailed mechanistic understanding and which can be manipulated by effective treatment strategies such as IPC.

Supplementary Figure Legend from Cancer Exosomes Trigger Fibroblast to Myofibroblast Differentiation

The kidney is the organ responsible for the regulation of the body’s fluid balance. Hyaluronan (HA), because of its unique hydration capacity, is used dynamically by the normal renal medulla to ensure whole-body fluid homeostasis. While levels of HA in the medulla fluctuate and can be very high, HA levels in the renal cortex remain static and are almost undetectable. Levels in the cortex are only upregulated in both acute and chronic renal diseases such as diabetes, ischemia-reperfusion injury, tubulointerstitial inflammation and renal transplant rejection. This chapter describes our current understanding of the mechanisms controlling the upregulation of HA in progressive disease, their potential recapitulation of embryonic events and which intracellular signalling pathways may have the potential to prevent or reverse the progression that leads to End Stage Renal Failure.

Chronic, non-healing wounds represent a significant area of unmet medical need and are a growing problem for healthcare systems around the world. They affect the quality of life for patients and are an economic burden, being difficult and time consuming to treat. They are an escalating problem across the developed world due to the increasing incidence of diabetes and the higher prevalence of ageing populations. Effective treatment options are currently lacking, and in some cases chronic wounds can persist for years. Some traditional medicines are believed to contain bioactive small molecules that induce the healing of chronic wounds by reducing excessive inflammation, thereby allowing re-epithelisation to occur. Furthermore, many small molecules found in plants are known to have antibacterial properties and, although they lack the therapeutic selectivity of antibiotics, they are certainly capable of acting as topical antiseptics when applied to infected wounds. As these molecules act through mechanisms of action distinct from those of clinically used antibiotics, they are often active against antibiotic resistant bacteria. Although there are numerous studies highlighting the effects of naturally occurring small molecules in wound-healing assays in vitro, only evidence from well conducted clinical trials can allow these molecules or the remedies that contain them to progress to the clinic. With this in mind, we review wound-healing natural remedies that have entered clinical trials over a twenty-year period to the present. We examine the bioactive small molecules likely to be in involved and, where possible, their mechanisms of action.

ABSTRACT Acute Kidney Injury (AKI) is a common cause of Chronic Kidney Disease (CKD). The leading cause of AKI worldwide is Ischaemia Reperfusion Injury (IRI), seen most commonly in the clinical setting as a result of sepsis-driven hypotension. We are increasingly recognising, however, that AKI and CKD are one closely associated continuum of disease, rather than distinct entities. Ischaemic Preconditioning (IPC) is a strategy aimed at reducing the deleterious effects of IRI. This study demonstrates an efficacious model of kidney IRI, and the protective influence of IPC in attenuating renal injury/fibrosis. A rat model of bilateral kidney IRI was used: Male Lewis rats (n=84) were assigned to IRI, sham or IPC. In IRI, renal pedicles were clamped for 45 minutes. IPC groups underwent pulsatile IPC prior to IRI. Kidneys were retrieved at 24-hours, 48-hours, 7-days, 14-days and 28-days, and assessed histologically. IRI led to marked histological damage and renal fibrosis development by 28 days. Histological injury scores and degree of fibrosis were significantly increased following IRI and attenuated with IPC. IPC resulted in a 66% reduction in renal fibrosis at 28 days (p<0.001). IRI also led to a significant increase in serum creatinine acutely, which was attenuated by IPC (p<0.0001). Interestingly at 14-days, there was limited histological damage and differentiation between IRI and IPC kidneys was difficult. IPC can protect from both acute and chronic kidney damage. 14-days post IRI represents a transitional phase, which maybe a timepoint for commitment to either fibrosis or recovery, and hence offers potential for therapeutic intervention.

Bioactivity-guided fractionation was used to isolate two compounds, tomentosenol A (1) and torellianone A (2), from a cerumen extract from Tetragonula carbonaria. The anti-fibrotic activity of these compounds was examined using human cultured neonatal foreskin fibroblasts (NFF) and immortalised keratinocytes (HaCaTs). Tomentosenol A (1), inhibited NFF and HaCaT cell proliferation and prevented NFF and HaCaT scratch wound repopulation at 12.5–25 µM concentrations. These inhibitory effects were associated with reduced cell viability, determined by tetrazolium dye (MTT) and sulforhodamine B (SRB) assays. Compound 1 further inhibited transforming growth factor-β1 (TGF-β1)-stimulated, NFF-myofibroblast differentiation and soluble collagen production; and was an effective scavenger of the model oxidant, 2,2-diphenyl-1-picrylhydrazyl (DPPH·), with an EC50 value of 44.7 ± 3.1 µM. These findings reveal significant anti-fibrotic potential for cerumen-derived tomentosenol A (1).

Myofibroblasts are contractile, α-smooth muscle actin-positive cells with multiple roles in pathophysiological processes. Myofibroblasts mediate wound contractions, but their persistent presence in tissues is central to driving fibrosis, making them attractive cell targets for the development of therapeutic treatments. However, due to shared cellular markers with several other phenotypes, the specific targeting of myofibroblasts has long presented a scientific and clinical challenge. In recent years, myofibroblasts have drawn much attention among scientific research communities from multiple disciplines and specialisations. As further research uncovers the characterisations of myofibroblast formation, function, and regulation, the realisation of novel interventional routes for myofibroblasts within pathologies has emerged. The research community is approaching the means to finally target these cells, to prevent fibrosis, accelerate scarless wound healing, and attenuate associated disease-processes in clinical settings. This comprehensive review article describes the myofibroblast cell phenotype, their origins, and their diverse physiological and pathological functionality. Special attention has been given to mechanisms and molecular pathways governing myofibroblast differentiation, and updates in molecular interventions.

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-β1 and proinflammatory interleukin (IL)-1β are widely associated with fibrotic progression. TGF-β1 induces myofibroblast differentiation, while IL-1β induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-β1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-β1- and IL-1β-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-β1-induced fibroblast/myofibroblast differentiation and IL-1β-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell-cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.

Fibroblasts are central to wound healing and fibrosis through Transforming Growth Factor- β 1 (TGF- β 1)-triggered differentiation into contractile, α -smooth muscle actin ( α -sma)-positive myofibroblasts. This is mediated by accumulation of a pericellular matrix of hyaluronan (HA) and the HA-dependent co-localisation of CD44 with the Epidermal Growth Factor Receptor (EGFR). Interactions of HA with hyaladherins, such as Inter-alpha-Inhibitor (IaI) and Tumour Necrosis Factor-stimulated gene-6 (TSG-6) are also essential for differentiation. This study investigated the mechanisms involved. TSG-6 and α -sma had different kinetics of induction by TGF-β 1, with TSG-6 peaking before α -sma. siCD44 or EGFR

Epoxy-tiglianes are a novel class of diterpene esters. The prototype epoxy-tigliane, EBC-46 (tigilanol tiglate), possesses potent anti-cancer properties and is currently in clinical development as a local treatment for human and veterinary cutaneous tumors. EBC-46 rapidly destroys treated tumors and consistently promotes wound reepithelialization at sites of tumor destruction. However, the mechanisms underlying these keratinocyte wound healing responses are not completely understood. Here, we investigated the effects of EBC-46 and an analogue (EBC-211) at 1.51 nM-151 mu M concentrations, on wound healing responses in immortalized human skin keratinocytes (HaCaTs). Both EBC-46 and EBC-211 (1.51 nM-15.1 mu M) accelerated G0/G1-S and S-G2/M cell cycle transitions and HaCaT proliferation. EBC-46 (1.51-151 nM) and EBC-211 (1.51 nM-15.1 mu M) further induced significant HaCaT migration and scratch wound repopulation. Stimulated migration/wound repopulation responses were even induced by EBC-46 (1.51 nM) and EBC-211 (1.51-151 nM) with proliferation inhibitor, mitomycin C (1 mu M), suggesting that epoxy-tiglianes can promote migration and wound repopulation independently of proliferation. Expression profiling analyses showed that epoxy-tiglianes modulated keratin, DNA synthesis/replication, cell cycle/proliferation, motility/migration, differentiation, matrix metalloproteinase (MMP) and cytokine/chemokine gene expression, to facilitate enhanced responses. Although epoxy-tiglianes down-regulated established cytokine and chemokine agonists of keratinocyte proliferation and migration, enhanced HaCaT responses were demonstrated to be mediated via protein kinase C (PKC) phosphorylation and significantly abrogated by pan-PKC inhibitor, bisindolylmaleimide-1 (BIM-1, 1 mu M). By identifying how epoxytiglianes stimulate keratinocyte healing responses and re-epithelialization in treated skin, our findings support the further development of this class of small molecules as potential therapeutics for other clinical situations associated with impaired re-epithelialization, such as non-healing skin wounds.

Hyaluronidase (HYAL)-2 is a weak, acid-active, hyaluronan-degrading enzyme broadly expressed in somatic tissues. Aberrant HYAL2 expression is implicated in diverse pathology. However, a significant proportion of HYAL2 is enzymatically inactive; thus the mechanisms through which HYAL2 dysregulation influences pathobiology are unclear. Recently, nonenzymatic HYAL2 functions have been described, and nuclear HYAL2 has been shown to influence mRNA splicing to prevent myofibroblast differentiation. Myofibroblasts drive fibrosis, thereby promoting progressive tissue damage and leading to multimorbidity. This study identifies a novel HYAL2 cytoplasmic function in myofibroblasts that is unrelated to its enzymatic activity. In fibroblasts and myofibroblasts, HYAL2 interacts with the GTPase-signaling small molecule ras homolog family member A (RhoA). Transforming growth factor beta 1-driven fibroblast-to-myofibroblast differentiation promotes HYAL2 cytoplasmic relocalization to bind to the actin cytoskeleton. Cytoskeletal-bound HYAL2 functions as a key regulator of downstream RhoA signaling and influences profibrotic myofibroblast functions, including myosin light-chain kinase-mediated myofibroblast contractility, myofibroblast migration, myofibroblast collagen/fibronectin deposition, as well as connective tissue growth factor and matrix metalloproteinase-2 expression. These data demonstrate that, in certain biological contexts, the nonenzymatic effects of HYAL2 are crucial in orchestrating RhoA signaling and downstream pathways that are important for full profibrotic myofibroblast functionality. In conjunction with previous data demonstrating the influence of HYAL2 on RNA splicing, these findings begin to explain the broad biological effects of HYAL2.

Hyaluronan (HA), an extra-cellular matrix glycosaminoglycan, may play a role in mesenchymal stem cell differentiation to fat but results using murine models and cell lines are conflicting. Our previous data, illustrating decreased HA production during human adipogenesis, suggested an inhibitory role. We have investigated the role of HA in adipogenesis and fat accumulation using human primary subcutaneous preadipocyte/fibroblasts (PFs, n = 12) and subjects of varying body mass index (BMI). The impact of HA on peroxisome proliferator-activated receptor gamma (PPARγ) expression was analysed following siRNA knockdown or HA synthase (HAS)1 and HAS2 overexpression. PFs were cultured in complete or adipogenic medium (ADM) with/without 4-methylumbelliferone (4-MU = HA synthesis inhibitor). Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, and measurement of a terminal differentiation marker. Modulating HA production by HAS2 knockdown or overexpression increased (16%, p < 0.04) or decreased (30%, p = 0.01) PPARγ transcripts respectively. The inhibition of HA by 4-MU significantly enhanced ADM-induced adipogenesis with 1.52 ± 0.18- (ORO), 4.09 ± 0.63- (foci) and 2.6 ± 0.21-(marker)-fold increases compared with the controls, also increased PPARγ protein expression (40%, (p < 0.04)). In human subjects, circulating HA correlated negatively with BMI and triglycerides (r = −0.396 (p = 0.002), r = −0.269 (p = 0.038), respectively), confirming an inhibitory role of HA in human adipogenesis. Thus, enhancing HA action may provide a therapeutic target in obesity.