Multiscale computational model predicts how environmental changes and drug treatments affect microvascular remodeling in fibrotic disease.

Investigating the molecular, cellular, and tissue-level changes caused by disease, and the effects of pharmacological treatments across these biological scales, necessitates the use of multiscale computational modeling in combination with experimentation. Many diseases dynamically alter the tissue microenvironment in ways that trigger microvascular network remodeling, which leads to the expansion or regression of microvessel networks. When microvessels undergo remodeling in idiopathic pulmonary fibrosis (IPF), functional gas exchange is impaired due to loss of alveolar structures and lung function declines. Here, we integrated a multiscale computational model with independent experiments to investigate how combinations of biomechanical and biochemical cues in IPF alter cell fate decisions leading to microvascular remodeling. Our computational model predicted that extracellular matrix (ECM) stiffening reduced microvessel area, which was accompanied by physical uncoupling of endothelial cell (ECs) and pericytes, the cells that comprise microvessels. Nintedanib, an FDA-approved drug for treating IPF, was predicted to further potentiate microvessel regression by decreasing the percentage of quiescent pericytes while increasing the percentage of pericytes undergoing pericyte-myofibroblast transition (PMT) in high ECM stiffnesses. Importantly, the model suggested that YAP/TAZ inhibition may overcome the deleterious effects of nintedanib by promoting EC-pericyte coupling and maintaining microvessel homeostasis. Overall, our combination of computational and experimental modeling can explain how cell decisions affect tissue changes during disease and in response to treatments.

International Delphi Consensus on the Management of AQP4-IgG+ NMOSD: Recommendations for Eculizumab, Inebilizumab, and Satralizumab.

BACKGROUND AND OBJECTIVES: Neuromyelitis optica spectrum disorder (NMOSD) is a rare debilitating autoimmune disease of the CNS. Three monoclonal antibodies were recently approved as maintenance therapies for aquaporin-4 immunoglobulin G (AQP4-IgG)-seropositive NMOSD (eculizumab, inebilizumab, and satralizumab), prompting the need to consider best practice therapeutic decision-making for this indication. Our objective was to develop validated statements for the management of AQP4-IgG-seropositive NMOSD, through an evidence-based Delphi consensus process, with a focus on recommendations for eculizumab, inebilizumab, and satralizumab. METHODS: We recruited an international panel of clinical experts in NMOSD and asked them to complete a questionnaire on NMOSD management. Panel members received a summary of evidence identified through a targeted literature review and provided free-text responses to the questionnaire based on both the data provided and their clinical experience. Responses were used to generate draft statements on NMOSD-related themes. Statements were voted on over a maximum of 3 rounds; participation in at least 1 of the first 2 rounds was mandatory. Panel members anonymously provided their level of agreement (6-point Likert scale) on each statement. Statements that failed to reach a predefined consensus threshold (≥67%) were revised based on feedback and then voted on in the next round. Final statements were those that met the consensus threshold (≥67%). RESULTS: The Delphi panel comprised 24 experts, who completed the Delphi process in November 2021 after 2 voting rounds. In round 1, 23/25 statements reached consensus and were accepted as final. The 2 statements that failed to reach consensus were revised. In round 2, both revised statements reached consensus. Twenty-five statements were agreed in total: 11 on initiation of or switching between eculizumab, inebilizumab, and satralizumab; 3 on monotherapy/combination therapy; 7 on safety and patient population considerations; 3 on biomarkers/patient-reported outcomes; and 1 on research gaps. DISCUSSION: An established consensus method was used to develop statements relevant to the management of AQP4-IgG-seropositive NMOSD. These international statements will be valuable for informing individualized therapeutic decision-making and could form the basis for standardized practice guidelines.

Variations in mechanical stiffness alter microvascular sprouting and stability in a PEG hydrogel model of idiopathic pulmonary fibrosis.

OBJECTIVE: Microvascular remodeling is governed by biomechanical and biochemical cues which are dysregulated in idiopathic pulmonary fibrosis. Understanding how these cues impact endothelial cell-pericyte interactions necessitates a model system in which both variables can be independently and reproducibly modulated. In this study we develop a tunable hydrogel-based angiogenesis assay to study how varying angiogenic growth factors and environmental stiffness affect sprouting and vessel organization. METHODS: Lungs harvested from mice were cut into 1 mm long segments then cultured on hydrogels having one of seven possible stiffness and growth factor combinations. Time course, brightfield, and immunofluorescence imaging were used to observe and quantify sprout formation. RESULTS: Our assay was able to support angiogenesis in a comparable manner to Matrigel in soft 2 kPa gels while enabling tunability to study the effects of stiffness on sprout formation. Matrigel and 2 kPa groups contained significantly more samples with sprouts when compared to the stiffer 10 and 20 kPa gels. Growth factor treatment did not have as obvious an effect, although the 20 kPa PDGF + FGF-treated group had significantly longer vessels than the vascular endothelial growth factor-treated group. CONCLUSIONS: We have developed a novel, tunable hydrogel assay for the creation of lung explant vessel organoids which can be modulated to study the impact of specific environmental cues on vessel formation and maturation.

Effect of Ocrelizumab on B- and T-Cell Receptor Repertoire Diversity in Patients With Relapsing Multiple Sclerosis From the Randomized Phase III OPERA Trial.

BACKGROUND AND OBJECTIVES: The B cell-depleting anti-CD20 antibody ocrelizumab (OCR) effectively reduces MS disease activity and slows disability progression. Given the role of B cells as antigen-presenting cells, the primary goal of this study was to evaluate the effect of OCR on the T-cell receptor repertoire diversity. METHODS: To examine whether OCR substantially alters the molecular diversity of the T-cell receptor repertoire, deep immune repertoire sequencing (RepSeq) of CD4+ and CD8+ T-cell receptor ß-chain variable regions was performed on longitudinal blood samples. The IgM and IgG heavy chain variable region repertoire was also analyzed to characterize the residual B-cell repertoire under OCR treatment. RESULTS: Peripheral blood samples for RepSeq were obtained from 8 patients with relapsing MS enrolled in the OPERA I trial over a period of up to 39 months. Four patients each were treated with OCR or interferon ß1-a during the double-blind period of OPERA I. All patients received OCR during the open-label extension. The diversity of the CD4+/CD8+ T-cell repertoires remained unaffected in OCR-treated patients. The expected OCR-associated B-cell depletion was mirrored by reduced B-cell receptor diversity in peripheral blood and a shift in immunoglobulin gene usage. Despite deep B-cell depletion, longitudinal persistence of clonally related B-cells was observed. DISCUSSION: Our data illustrate that the diversity of CD4+/CD8+ T-cell receptor repertoires remained unaltered in OCR-treated patients with relapsing MS. Persistence of a highly diverse T-cell repertoire suggests that aspects of adaptive immunity remain intact despite extended anti-CD20 therapy. TRIAL REGISTRATION INFORMATION: This is a substudy (BE29353) of the OPERA I (WA21092; NCT01247324) trial. Date of registration, November 23, 2010; first patient enrollment, August 31, 2011.

Remote Observational Research for Multiple Sclerosis: A Natural Experiment.

BACKGROUND AND OBJECTIVES: Prospective, deeply phenotyped research cohorts monitoring individuals with chronic neurologic conditions, such as multiple sclerosis (MS), depend on continued participant engagement. The COVID-19 pandemic restricted in-clinic research activities, threatening this longitudinal engagement, but also forced adoption of televideo-enabled care. This offered a natural experiment in which to analyze key dimensions of remote research: (1) comparison of remote vs in-clinic visit costs from multiple perspectives and (2) comparison of the remote with in-clinic measures in cross-sectional and longitudinal disability evaluations. METHODS: Between March 2020 and December 2021, 207 MS cohort participants underwent hybrid in-clinic and virtual research visits; 96 contributed 100 "matched visits," that is, in-clinic (Neurostatus-Expanded Disability Status Scale [NS-EDSS]) and remote (televideo-enabled EDSS [tele-EDSS]; electronic patient-reported EDSS [ePR-EDSS]) evaluations. Clinical, demographic, and socioeconomic characteristics of participants were collected. RESULTS: The costs of remote visits were lower than in-clinic visits for research investigators (facilities, personnel, parking, participant compensation) but also for participants (travel, caregiver time) and carbon footprint (p < 0.05 for each). Median cohort EDSS was similar between the 3 modalities (NS-EDSS: 2, tele-EDSS: 1.5, ePR-EDSS: 2, range 0.6.5); the remote evaluations were each noninferior to the NS-EDSS within ±0.5 EDSS point (TOST for noninferiority, p < 0.01 for each). Furthermore, year to year, the % of participants with worsening/stable/improved EDSS scores was similar, whether each annual evaluation used NS-EDSS or whether it switched from NS-EDSS to tele-EDSS. DISCUSSION: Altogether, the current findings suggest that remote evaluations can reduce the costs of research participation for patients, while providing a reasonable evaluation of disability trajectory longitudinally. This could inform the design of remote research that is more inclusive of diverse participants.

Secondary Progressive Multiple Sclerosis: A Review of Clinical Characteristics, Definition, Prognostic Tools, and Disease-Modifying Therapies.

Many challenges exist in the precise diagnosis and clinical management of secondary progressive multiple sclerosis (SPMS) because of the lack of definitive clinical, imaging, immunologic, or pathologic criteria that demarcate the transition from relapsing-remitting MS to SPMS. This review provides an overview of the diagnostic criteria/definition and the heterogeneity associated with different SPMS patient populations; it also emphasizes the importance of available prospective/retrospective tools to identify patients with SPMS earlier in the disease course so that approved disease-modifying therapies and nonpharmacological strategies will translate into better outcomes. Delivery of such interventions necessitates an evolving patient-clinician dialog within the context of a multidisciplinary team.

Photoacoustic microscopy of vascular adaptation and tissue oxygen metabolism during cutaneous wound healing.

Cutaneous wounds affect millions of people every year. Vascularization and blood oxygen delivery are critical bottlenecks in wound healing, and understanding the spatiotemporal dynamics of these processes may lead to more effective therapeutic strategies to accelerate wound healing. In this work, we applied multi-parametric photoacoustic microscopy (PAM) to study vascular adaptation and the associated changes in blood oxygen delivery and tissue oxygen metabolism throughout the hemostasis, inflammatory, proliferation, and early remodeling phases of wound healing in mice with skin puncture wounds. Multifaceted changes in the vascular structure, function, and tissue oxygen metabolism were observed during the 14-day monitoring of wound healing. On the entire wound area, significant elevations of the arterial blood flow and tissue oxygen metabolism were observed right after wounding and remained well above the baseline over the 14-day period. On the healing front, biphasic changes in the vascular density and blood flow were observed, both of which peaked on day 1, remained elevated in the first week, and returned to the baselines by day 14. Along with the wound closure and thickening, tissue oxygen metabolism in the healing front remained elevated even after structural and functional changes in the vasculature were stabilized. On the newly formed tissue, significantly higher blood oxygenation, flow, and tissue metabolism were observed compared to those before wounding. Blood oxygenation and flow in the new tissue appeared to be independent of when it was formed, but instead showed noticeable dependence on the phase of wound healing. This PAM study provides new insights into the structural, functional, and metabolic changes associated with vascular adaptation during wound healing and suggests that the timing and target of vascular treatments for wound healing may affect the outcomes.

Immunomodulation of Acellular Dermal Matrix Through Interleukin 4 Enhances Vascular Infiltration.

BACKGROUND: Acellular dermal matrix (ADM) supported implant-based reconstruction remains the most commonly performed mode of reconstruction after breast cancer. Acellular dermal matrix clinical usage has reported benefits but requires rapid and efficient vascular and cellular incorporation into the recipient to have the best outcomes. Orderly transition from M1 to M2 macrophage phenotypic profile, coordinated in part by interleukin 4 (IL-4), is an important component of vascular stabilization and remodeling. Using the ADM substrate as a delivery device for immunomodulation of macrophage phenotype holds the potential to improve integration. METHODS: Interleukin 4 was adsorbed onto ADM samples and drug elution curves were measured. Next, experimental groups of 8 C57BL/6 mice had 5-mm ADM discs surgically placed in a dorsal window chamber with a vascularized skin flap on one side and a plastic cover slip on the other in a model of implant-based breast reconstruction. Group 1 consisted of IL-4 (5 µg) adsorbed into the ADM preoperatively and group 2 consisted of an untreated ADM control. Serial gross examinations were performed with histology at day 21 for markers of vascularization, mesenchymal cell infiltration, and macrophage lineage. RESULTS: Drug elution curves showed sustained IL-4 release for 10 days after adsorption. Serial gross examination showed similar rates of superficial vascular investment of the ADM beginning at the periphery by day 14 and increasing through day 21. Interleukin-4 treatment led to significantly increased CD31 staining of vascular endothelial cells within the ADM over the control group (P < 0.05) at 21 days. Although vimentin staining did not indicate a significant increase in fibroblasts overall, IL-4 did result in a significant increase in expression of α-smooth muscle actin. The expression of macrophage phenotype markers Arginase1 and iNOS present within the ADM were not significantly affected by IL-4 treatment at the day 21 time point. CONCLUSIONS: Acellular dermal matrix has the potential to be used for immunomodulatory cytokine delivery during the timeframe of healing. Using implanted ADM as a delivery vehicle to drive IL-4 mediated angiogenesis and vascular remodeling significantly enhanced vascularity within the ADM substrate.

COVID-19 Infection in Fingolimod- or Siponimod-Treated Patients: Case Series.

BACKGROUND AND OBJECTIVES: A descriptive analysis of COVID-19 infection in patients with multiple sclerosis (MS) receiving fingolimod or siponimod. METHODS: We reviewed the cases of COVID-19 from postmarketing or ongoing clinical trials reported to Novartis through December 27, 2020. RESULTS: As of December 27, 2020, 283 cases had been reported in fingolimod-treated patients. The mean age was 44 years (from n = 224; range 11-69 years), and 190 were women. Of 161 cases with available information, 138 were asymptomatic (6), mild (100), or moderate (32); 50 cases required hospitalization. At the last follow-up, 140 patients were reported as recovered/recovering, condition was unchanged in 22, and deteriorated in 3 patients; 4 patients had a fatal outcome. Information was not available for 114 patients. Of the 54 cases of COVID-19 reported in siponimod-treated patients, 45 were from the postmarketing setting and 9 from an ongoing open-label clinical trial. The mean age was 54 years (from n = 45; range 31-70), and 30 were women. Of 28 cases with available information, 24 were asymptomatic (2), mild (17), or moderate (5); 9 cases required hospitalization. At the last follow-up, 27 patients were reported as recovered/recovering, condition remained unchanged for 1, and 3 patients had a fatal outcome. Information was not available for 23 patients. DISCUSSION: Based on a review of available information, the risk of more severe COVID-19 in patients receiving fingolimod or siponimod seems to be similar to that reported in the general population and the MS population with COVID-19. However, limitations of spontaneous reporting, especially missing data, should be considered in the interpretation of these observations.

Mathematical Model Predicts that Acceleration of Diabetic Wound Healing is Dependent on Spatial Distribution of VEGF-A mRNA (AZD8601).

INTRODUCTION: Pharmacologic approaches for promoting angiogenesis have been utilized to accelerate healing of chronic wounds in diabetic patients with varying degrees of success. We hypothesize that the distribution of proangiogenic drugs in the wound area critically impacts the rate of closure of diabetic wounds. To evaluate this hypothesis, we developed a mathematical model that predicts how spatial distribution of VEGF-A produced by delivery of a modified mRNA (AZD8601) accelerates diabetic wound healing. METHODS: We modified a previously published model of cutaneous wound healing based on coupled partial differential equations that describe the density of sprouting capillary tips, chemoattractant concentration, and density of blood vessels in a circular wound. Key model parameters identified by a sensitivity analysis were fit to data obtained from an in vivo wound healing study performed in the dorsum of diabetic mice, and a pharmacokinetic model was used to simulate mRNA and VEGF-A distribution following injections with AZD8601. Due to the limited availability of data regarding the spatial distribution of AZD8601 in the wound bed, we performed simulations with perturbations to the location of injections and diffusion coefficient of mRNA to understand the impact of these spatial parameters on wound healing. RESULTS: When simulating injections delivered at the wound border, the model predicted that injections delivered on day 0 were more effective in accelerating wound healing than injections delivered at later time points. When the location of the injection was varied throughout the wound space, the model predicted that healing could be accelerated by delivering injections a distance of 1-2 mm inside the wound bed when compared to injections delivered on the same day at the wound border. Perturbations to the diffusivity of mRNA predicted that restricting diffusion of mRNA delayed wound healing by creating an accumulation of VEGF-A at the wound border. Alternatively, a high mRNA diffusivity had no effect on wound healing compared to a simulation with vehicle injection due to the rapid loss of mRNA at the wound border to surrounding tissue. CONCLUSIONS: These findings highlight the critical need to consider the location of drug delivery and diffusivity of the drug, parameters not typically explored in pre-clinical experiments, when designing and testing drugs for treating diabetic wounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00678-9.

Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis.

Retinal diseases are frequently characterized by the accumulation of excessive scar tissue found throughout the neural retina. However, the pathophysiology of retinal fibrosis remains poorly understood, and the cell types that contribute to the fibrotic response are incompletely defined. Here, we show that myofibroblast differentiation of mural cells contributes directly to retinal fibrosis. Using lineage tracing technology, we demonstrate that after chemical ocular injury, Myh11+ mural cells detach from the retinal microvasculature and differentiate into myofibroblasts to form an epiretinal membrane. Inhibition of TGFßR attenuates Myh11+ retinal mural cell myofibroblast differentiation, and diminishes the subsequent formation of scar tissue on the surface of the retina. We demonstrate retinal fibrosis within a murine model of oxygen-induced retinopathy resulting from the intravitreal injection of adipose Myh11-derived mesenchymal stem cells, with ensuing myofibroblast differentiation. In this model, inhibiting TGFßR signaling does not significantly alter myofibroblast differentiation and collagen secretion within the retina. This work shows the complexity of retinal fibrosis, where scar formation is regulated both by TGFßR and non-TGFßR dependent processes involving mural cells and derived mesenchymal stem cells. It also offers a cautionary note on the potential deleterious, pro-fibrotic effects of exogenous MSCs once intravitreally injected into clinical patients.

Oxygen-Sensing Biomaterial Construct for Clinical Monitoring of Wound Healing.

OBJECTIVE: Oxygen is essential to wound healing; therefore, accurate monitoring can guide clinical decisions. Clinical wound assessment is often subjective, and tools to monitor wound oxygen are typically expensive, indirect, and highly variable. This study demonstrates the utility of a novel, low-cost oxygen-sensing thin film for serial assessment of wound oxygenation. DESIGN: Dual-layer films were fabricated with boron oxygen-sensing nanoparticles (BNPs) impregnated into a chitosan-polycaprolactone layer for direct wound bed contact with a relatively oxygen impermeable calcium alginate surface layer. The BNPs are a dual-emissive difluoroboron ß-diketonate dye incorporated into poly(lactic acid) nanoparticles. Under UV excitation, the BNPs emit fluorescence based on concentration and oxygen-sensitive phosphorescence. The fluorescence/phosphorescence ratio is directly proportional to oxygen concentration. METHODS: A series of in vitro oxygen challenges and in vivo murine and porcine wound healing models were used to validate the utility of the film in sensing wound oxygenation. MAIN RESULTS: In vitro testing demonstrated the oxygen-sensing capability of the BNP film and its ability to shield ambient oxygen to isolate wound oxygen. In vivo testing demonstrated the ability of the film to accurately monitor relative oxygen changes in a murine wound over time, measuring a 22% fluorescence/phosphorescence increase during acute healing. CONCLUSIONS: This study presents a low-cost, noninvasive, direct, and serial oxygen mapping technology to detect spatial differences in wound oxygenation. Clinical use of the films has the potential to monitor wound healing trajectories and guide wound care decisions.

Model-Based Analysis Reveals a Sustained and Dose-Dependent Acceleration of Wound Healing by VEGF-A mRNA (AZD8601).

Intradermal delivery of AZD8601, an mRNA designed to produce vascular endothelial growth factor A (VEGF-A), has previously been shown to accelerate cutaneous wound healing in a murine diabetic model. Here, we develop population pharmacokinetic and pharmacodynamic models aiming to quantify the effect of AZD8601 injections on the dynamics of wound healing. A dataset of 584 open wound area measurements from 131 mice was integrated from 3 independent studies encompassing different doses, dosing timepoints, and number of doses. Evaluation of several candidate models showed that wound healing acceleration is not likely driven directly by time-dependent VEGF-A concentration. Instead, we found that administration of AZD8601 induced a sustained acceleration of wound healing depending on the accumulated dose, with a dose producing 50% of the maximal effect of 92 µg. Simulations with this model showed that a single dose of 200 µg AZD8601 can reduce the time to reach 50% wound healing by up to 5 days.

Innovations hair restoration surgeons have made to adapt to the challenges of follicular unit excision.

BACKGROUND: Early use of follicular unit excision (FUE) as a method of hair transplantation was limited by high rates of hair follicle transection. This hurdle has been overcome by innovative methods, punch shapes, and devices. With the vast array of tools available, it can be difficult for hair transplant surgeons to choose the best option for their practices. AIMS: To provide an in-depth review and comparison of currently available FUE methods, punch designs, and motorized devices, and discuss how these tools fit the unique skin and hair characteristics of patients. METHODS: A review of the literature and available information on FUE methods, punches, and devices, as well as the authors' experience in this area, is provided. RESULTS: Innovative FUE methods, punch shapes, and motorized devices have successfully minimized the rate of hair follicle transection. Methods include the use of sharp punches with depth control, and blunt rotating punches. Punch shapes such as flared, hybrid, and edge out have successfully reduced transections by keeping the cutting edge away from the follicles under the skin. The development of motorized devices using features including rotation, roto-oscillation, oscillation, vibration, suction, and hydration has also aided in achieving more successful graft excision. CONCLUSION: Follicular unit excision is a widely used technique by hair restoration surgeons. Therefore, it is important for physicians to be aware of the array of punches and devices available and understand how these tools can be used to adapt to the unique skin and hair characteristics of individual patients to optimize successful graft harvesting.

In vivo imaging of hemodynamic redistribution and arteriogenesis across microvascular network.

OBJECTIVE: Arteriogenesis is an important mechanism that contributes to restoration of oxygen supply in chronically ischemic tissues, but remains incompletely understood due to technical limitations. This study presents a novel approach for comprehensive assessment of the remodeling pattern in a complex microvascular network containing multiple collateral microvessels. METHODS: We have developed a hardware-software integrated platform for quantitative, longitudinal, and label-free imaging of network-wide hemodynamic changes and arteriogenesis at the single-vessel level. By ligating feeding arteries in the mouse ear, we induced network-wide hemodynamic redistribution and localized arteriogenesis. The utility of this technology was demonstrated by studying the influence of obesity on microvascular arteriogenesis. RESULTS: Simultaneously monitoring the remodeling of competing collateral arterioles revealed a new, inverse relationship between initial vascular resistance and extent of arteriogenesis. Obese mice exhibited similar remodeling responses to lean mice through the first week, including diameter increase and flow upregulation in collateral arterioles. However, these gains were subsequently lost in obese mice. CONCLUSIONS: Capable of label-free, comprehensive, and dynamic quantification of structural and functional changes in the microvascular network in vivo, this platform opens up new opportunities to study the mechanisms of microvascular arteriogenesis, its implications in diseases, and approaches to pharmacologically rectify microvascular dysfunction.

Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing.

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.

The miR-200b/200a/429 cluster prevents metastasis and induces dormancy in a murine claudin-low mammary tumor cell line.

The miR-200 family of microRNAs consisting of miR-141, miR-200a, miR-200b, miR-200c and miR-429 are emerging as important regulators of breast cancer progression. This family of microRNAs maintain mammary epithelial identity and downregulation of miR-200 expression has been associated with epithelial-to-mesenchymal transition in mammary tumors. Therefore, re-expression of one or more miR-200 family members in mammary tumor cells with mesenchymal characteristics may restore an epithelial phenotype including growth and metastasis suppression. To test this hypothesis, the miR-200b/200a/429 cluster was re-expressed in a murine claudin-low cell line, RJ423. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed the expression of Vim, Snai1, Twist1, Twist2 and Zeb1, reverted RJ423 cells to a more epithelial morphology and significantly inhibited proliferation in vitro. Moreover, the miR-200b/200a/429 cluster prevented lung metastasis in an experimental metastasis model and although tumor initiation was not prevented, re-expression of the miR-200b/200a/429 cluster induced a dormancy-like state where mammary tumors failed to grow beyond ~150 mm3 or grew extremely slowly following intra-mammary injection. These dormant tumors contained elevated levels of collagen and were highly vascularized. Therefore, re-expression of the miR-200b/200a/429 cluster in the claudin-low mammary tumor cell line, RJ423, is sufficient to alter cell morphology, impair metastasis and induce tumor dormancy.