Functional Nanocomposites in the Development of Flexible Armor.

The idea of flexible body armor has been around for many years. Initial development included shear thickening fluid (STF) as a backbone polymer to impregnate ballistic fibers such as Kevlar. At the core of the ballistic and spike resistance was the instantaneous rise in viscosity of STF during impact. Increase in viscosity was due to the hydroclustering of silica nanoparticles dispersed in polyethylene glycol (PEG) through a centrifuge and evaporation process. When STF composite was dry, hydroclustering was not possible due to absence of any fluidity in PEG. However, particles embedded within the polymer, covered the Kevlar fiber and offered some resistance to spike and ballistic penetration. The resistance was meagre and hence, the goal was to improve it further. This was achieved by creating chemical bonds between particles, and by strongly attaching particles to the fiber. PEG was replaced with silane (3-amino propyl trimethoxysilane), and a fixative cross-linker, Glutaraldehyde (Gluta), was added. Silane installed an amine functional group on the silica nanoparticle surface, and Gluta created strong bridges between distant pairs of amine groups. Amide functional groups present in Kevlar also interacted with Gluta and silane to form a secondary amine, allowing silica particles to attach to fiber. A network of amine bonding was also established across the particle-polymer-fiber system. In synthesizing the armor, silica nanoparticles were dispersed in a mixture of silane, ethanol, water, and Gluta, maintaining an appropriate ratio by weight, and using a sonication technique. Ethanol was used as a dispersion fluid and was evaporated later. Several layers of Kevlar fabric were then soaked with the admixture for about 24 h and dried in an oven. Armor composites were tested in a drop tower according to NIJ115 Standard using spikes. Kinetic energy at impact was calculated and normalized with the aerial density of the armor. NIJ tests revealed that normalized energy for 0-layer penetration increased from 10 J-cm2/g (STF composite) to 220 J-cm2/g for the new armor composite, indicating a 22-fold enhancement. SEM and FTIR studies confirmed that this high resistance to spike penetration was due to the formation of stronger C-N, C-H, and C=C-H stretches facilitated by the presence of silane and Gluta.

Design, Synthesis, and Evaluation of Novel Pyruvate Dehydrogenase Kinase Inhibitors.

AIMS: The present work describes the synthesis and the biological evaluation of novel compounds acting as pyruvate dehydrogenase kinase (PDK) inhibitors. These drugs should become a new therapeutic approach for the treatment of pathologies improved by the control of the blood lactate level. METHODS: Four series of compounds belonging to N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2- methylpropanamides and 1,2,4-benzothiadiazine 1,1-dioxides were prepared and evaluated as PDK inhibitors. RESULTS: The newly synthesized N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2-methylpropanamides structurally related to previously reported reference compounds 4 and 5 were found to be potent PDK inhibitors (i.e. 10d: IC50 = 41 nM). 1,2,4-Benzothiadiazine 1,1-dioxides carrying a (methyl/ trifluoromethyl)-propanamide moiety at the 6-position were also designed as conformationally restricted ring-closed analogues of N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2-hydroxy-2-methylpropanamides. Most of them were found to be less potent than their ring-opened analogues. Interestingly, the best choice of hydrocarbon side chain at the 4-position was the benzyl chain, providing 11c (IC50 = 3.6 µM) belonging to "unsaturated" 1,2,4-benzothiadiazine 1,1-dioxides, and 12c (IC50 = 0.5 µM) belonging to "saturated' 1,2,4-benzothiadiazine 1,1-dioxides. CONCLUSION: This work showed that ring-closed analogues of N-(4-(N-alkyl/aralkylsulfamoyl) phenyl)- 2-hydroxy-2-methylpropanamides were less active as PDK inhibitors than their corresponding ring-opened analogues. However, the introduction of a bulkier substituent at the 4-position of the 1,2,4-benzothiadiazine 1,1-dioxide core structure, such as a benzyl or a phenethyl side chain, was allowed, opening the way to the design of new inhibitors with improved PDK inhibitory activity.

Intravitreal injection of anti-miRs against miR-142-3p reduces angiogenesis and microglia activation in a mouse model of laser-induced choroidal neovascularization.

Age-related macular degeneration (AMD) is a worldwide leading cause of blindness affecting individuals over 50 years old. The most aggressive form, wet AMD, is characterized by choroidal neovascularization (CNV) and inflammation involving microglia recruitment. By using a laser-induced CNV mouse model, we provide evidence for a key role played by miR-142-3p during CNV formation. MiR-142-3p was overexpressed in murine CNV lesions and its pharmacological inhibition decreased vascular and microglia densities by 46% and 30%, respectively. Consistently, miR-142-3p overexpression with mimics resulted in an increase of 136% and 126% of blood vessels and microglia recruitment. Interestingly, miR-142-3p expression was linked to the activation state of mouse microglia cells as determined by morphological analysis (cell solidity) through a computational method. In vitro, miR-142-3p overexpression in human microglia cells (HMC3) modulated microglia activation, as shown by CD68 levels. Interestingly, miR142-3p modulation also regulated the production of VEGF-A, the main pro-angiogenic factor. Together, these data strongly support the unprecedented importance of miR-142-3p-dependent vascular-inflammation axis during CNV progression, through microglia activation.

Pyruvate dehydrogenase kinase/lactate axis: a therapeutic target for neovascular age-related macular degeneration identified by metabolomics.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in aging populations. Here, we applied metabolomics to human sera of patients with nAMD during an active (exudative) phase of the pathology and found higher lactate levels and a shift in the lipoprotein profile (increased VLDL-LDL/HDL ratio). Similar metabolomics changes were detected in the sera of mice subjected to laser-induced choroidal neovascularization (CNV). In this experimental model, we provide evidence for two sites of lactate production: first, a local one in the injured eye, and second a systemic site associated with the recruitment of bone marrow-derived inflammatory cells. Mechanistically, lactate promotes the angiogenic response and M2-like macrophage accumulation in the eyes. The therapeutic potential of our findings is demonstrated by the pharmacological control of lactate levels through pyruvate dehydrogenase kinase (PDK) inhibition by dichloroacetic acid (DCA). Mice treated with DCA exhibited normalized lactate levels and lipoprotein profiles, and inhibited CNV formation. Collectively, our findings implicate the key role of the PDK/lactate axis in AMD pathogenesis and reveal that the regulation of PDK activity has potential therapeutic value in this ocular disease. The results indicate that the lipoprotein profile is a traceable pattern that is worth considering for patient follow-up. KEY MESSAGES: Lactate and lipoprotein profile are associated with the active phase of AMD and CNV development. Lactate is a relevant and functional metabolite correlated with AMD progression. Modulating lactate through pyruvate dehydrogenase kinase led to a decrease of CNV progression. Pyruvate dehydrogenase kinase is a new therapeutic target for neovascular AMD.

Evaluation of the stability of measles vaccine out of the cold chain under extended controlled temperature conditions.

Measles outbreaks occur periodically in remote and difficult to reach areas in countries such as the Democratic Republic of Congo. The possibility to keep measles vaccines at temperatures outside the cold chain for a limited period prior to administration would be an advantage for organizations such as Médecins Sans Frontières, which repeatedly respond to measles outbreaks in difficult contexts. Using stability data at 37 °C and 40 °C provided by Serum Institute of India Private Limited we applied the product release model for Extended Controlled Temperature Conditions (ECTC) to evaluate the possibility of an out of the cold chain excursion. Measles vaccine in the lyophilized form remains above the minimum required potency at the end of the shelf-life for up to 6 days at 37 °C or for 2 days at 40 °C. This evaluation supports the use of a monodose presentation of measles vaccine in ECTC. This could be an advantage for outbreak response in isolated and difficult to reach areas. However the operational advantages of this approach need to be established.

Typhoid fever outbreak in the Democratic Republic of Congo: Case control and ecological study.

During 2011 a large outbreak of typhoid fever affected an estimated 1430 people in Kikwit, Democratic Republic of Congo. The outbreak started in military camps in the city but then spread to the general population. This paper reports the results of an ecological analysis and a case-control study undertaken to examine water and other possible transmission pathways. Attack rates were determined for health areas and risk ratios were estimated with respect to spatial exposures. Approximately 15 months after the outbreak, demographic, environmental and exposure data were collected for 320 cases and 640 controls residing in the worst affected areas, using a structured interview questionnaire. Unadjusted and adjusted odds ratios were estimated. Complete data were available for 956 respondents. Residents of areas with water supplied via gravity on the mains network were at much greater risk of disease acquisition (risk ratio = 6.20, 95%CI 3.39-11.35) than residents of areas not supplied by this mains network. In the case control study, typhoid was found to be associated with ever using tap water from the municipal supply (OR = 4.29, 95% CI 2.20-8.38). Visible urine or faeces in the latrine was also associated with increased risk of typhoid and having chosen a water source because it is protected was negatively associated. Knowledge that washing hands can prevent typhoid fever, and stated habit of handwashing habits before cooking or after toileting was associated with increased risk of disease. However, observed associations between handwashing or plate-sharing with disease risk could very likely be due to recall bias. This outbreak of typhoid fever was strongly associated with drinking water from the municipal drinking water supply, based on the descriptive and analytic epidemiology and the finding of high levels of faecal contamination of drinking water. Future outbreaks of potentially waterborne disease need an integrated response that includes epidemiology and environmental microbiology during early stages of the outbreak.

High Proportions of Patients With Advanced HIV Are Antiretroviral Therapy Experienced: Hospitalization Outcomes From 2 Sub-Saharan African Sites.

Background: Human immunodeficiency virus (HIV) remains an important cause of hospitalization and death in low- and middle- income countries. Yet morbidity and in-hospital mortality patterns remain poorly characterized, with prior antiretroviral therapy (ART) exposure and treatment failure status largely unknown. Methods: We studied HIV-infected inpatients aged ≥13 years from cohorts in Kenya and the Democratic Republic of Congo (DRC), assessing clinical and demographic characteristics and hospitalization outcomes. Kenyan inpatients were prospectively enrolled during hospitalization; identical retrospective data were extracted for Congolese patients meeting the study criteria using routine medical information. Results: Among 338 HIV-infected patients in Kenya and 411 in DRC, 83.7% (95% confidence interval [CI], 79.4%-87.3%) and 97.3% (95% CI, 95.2%-98.5%), were admitted with advanced disease (defined as CD4 <200 cells/µL or World Health Organization stage 3/4 illness). Among inpatients with advanced HIV, 35.4% and 21.7% were ART-naive at admission. Patients under care had a median time of 44.1 (interquartile range [IQR], 18.4-90.5) months and 55.9 (IQR, 28.1-99.6) months on treatment; 17.2% (95% CI, 13.5%-21.6%) and 29.6% (95% CI, 25.4%-34.3%) died, 25.9% (95% CI, 16.0%-39.0%) and 22.5% (95% CI, 15.8%-31.0%) of these within 48 hours. Conclusions: Across 2 diverse clinical contexts in sub-Saharan Africa, advanced HIV inpatients were frequently admitted with low CD4 counts, often failing first-line ART. Earlier identification of treatment failure and rapid switching to second-line ART are needed.

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay.

Lymphangiogenesis, the formation of new lymphatic vessels, occurs in primary tumors and in draining lymph nodes leading to pre-metastatic niche formation. Reliable in vivo models are becoming instrumental for investigating alterations occurring in lymph nodes before tumor cell arrival. In this study, we demonstrate that B16F10 melanoma cell encapsulation in a biomaterial, and implantation in the mouse ear, prevents their rapid lymphatic spread observed when cells are directly injected in the ear. Vascular remodeling in lymph nodes was detected two weeks after sponge implantation, while their colonization by tumor cells occurred two weeks later. In this model, a huge lymphangiogenic response was induced in primary tumors and in pre-metastatic and metastatic lymph nodes. In control lymph nodes, lymphatic vessels were confined to the cortex. In contrast, an enlargement and expansion of lymphatic vessels towards paracortical and medullar areas occurred in pre-metastatic lymph nodes. We designed an original computerized-assisted quantification method to examine the lymphatic vessel structure and the spatial distribution. This new reliable and accurate model is suitable for in vivo studies of lymphangiogenesis, holds promise for unraveling the mechanisms underlying lymphatic metastases and pre-metastatic niche formation in lymph nodes, and will provide new tools for drug testing.

Localised transmission hotspots of a typhoid fever outbreak in the Democratic Republic of Congo.

INTRODUCTION: In a semi-urban setting in the Democratic Republic of Congo, this study aims to understand the dynamic of a typhoid fever (TF) outbreak and to assess: a) the existence of hot spots for TF transmission and b) the difference between typhoid cases identified within those hot spots and the general population in relation to socio-demographic characteristics, sanitation practice, and sources of drinking water. METHODS: This was a retrospective analysis of TF outbreaks in 2011 in Kikwit, DRC using microbiological analysis of water sources and a structured interview questionnaire. RESULTS: There were a total of 1430 reported TF cases. The outbreak's epidemic curve shows earliest and highest peak attack rates (AR) in three military camps located in Kikwit (Ebeya 3.2%; Ngubu 3.0%; and Nsinga 2.2%) compared to an average peak AR of 0.6% in other affected areas. A total 320 cases from the military camps and the high burden health areas were interviewed. Typhoid cases in the military camps shared a latrine with more than one family (P<0.02). All tap water sources in both the military camps and general population were found to be highly contaminated with faecal coliforms. CONCLUSION: The role of military camps in Kikwit as early hotspots of TF transmission was likely associated with lower sanitary and hygiene conditions. The proximity of camps to the general population might have been responsible for disseminating TF to the general population. Mapping of cases during an outbreak could be crucial to identify hot spots for transmission and institute corrective measures.

One size fits all? Standardised provision of care for survivors of sexual violence in conflict and post-conflict areas in the Democratic Republic of Congo.

BACKGROUND: Outcomes of sexual violence care programmes may vary according to the profile of survivors, type of violence suffered, and local context. Analysis of existing sexual violence care services could lead to their better adaptation to the local contexts. We therefore set out to compare the Médecins Sans Frontières sexual violence programmes in the Democratic Republic of Congo (DRC) in a zone of conflict (Masisi, North Kivu) and post-conflict (Niangara, Haut-Uélé). METHODS: A retrospective descriptive cohort study, using routine programmatic data from the MSF sexual violence programmes in Masisi and Niangara, DRC, for 2012. RESULTS: In Masisi, 491 survivors of sexual violence presented for care, compared to 180 in Niangara. Niangara saw predominantly sexual violence perpetrated by civilians who were known to the victim (48%) and directed against children and adolescents (median age 15 (IQR 13-17)), while sexual violence in Masisi was more directed towards adults (median age 26 (IQR 20-35)), and was characterised by marked brutality, with higher levels of gang rape, weapon use, and associated violence; perpetrated by the military (51%). Only 60% of the patients in Masisi and 32% of those in Niangara arrived for a consultation within the critical timeframe of 72 hours, when prophylaxis for HIV and sexually transmitted infections is most effective. Survivors were predominantly referred through community programmes. Treatment at first contact was typically efficient, with high (>95%) coverage rates of prophylaxes. However, follow-up was poor, with only 49% of all patients in Masisi and 61% in Niangara returning for follow-up, and consequently low rates of treatment and/or vaccination completion. CONCLUSION: This study has identified a number of weak and strong points in the sexual violence programmes of differing contexts, indicating gaps which need to be addressed, and strengths of both programmes that may contribute to future models of context-specific sexual violence programmes.

An easy, convenient cell and tissue extraction protocol for nuclear magnetic resonance metabolomics.

INTRODUCTION: As a complement to the classic metabolomics biofluid studies, the visualisation of the metabolites contained in cells or tissues could be a very powerful tool to understand how the local metabolism and biochemical pathways could be affected by external or internal stimuli or pathologies. Therefore, extraction and/or lysis is necessary to obtain samples adapted for use with the current analytical tools (liquid NMR and MS). These extraction or lysis work-ups are often the most labour-intensive and rate-limiting steps in metabolomics, as they require accuracy and repeatability as well as robustness. Many of the procedures described in the literature appear to be very time-consuming and not easily amenable to automation. OBJECTIVE: To find a fast, simplified procedure that allows release of the metabolites from cells and tissues in a way that is compatible with NMR analysis. METHODS: We assessed the use of sonication to disrupt cell membranes or tissue structures. Both a vibrating probe and an automated bath sonicator were explored. RESULTS: The application of sonication as the disruption procedure led to reproducible NMR spectral data compatible with metabolomics studies. This method requires only a small biological tissue or cell sample, and a rapid, reduced work-up was applied before analysis. The spectral patterns obtained are comparable with previous, well-described extraction protocols. CONCLUSION: The rapidity and the simplicity of this approach could represent a suitable alternative to the other protocols. Additionally, this approach could be favourable for high- throughput applications in intracellular and intratissular metabolite measurements.

Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice.

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruch's membrane, resulting in subretinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenoviruses and pre-microRNAs to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7-14 d to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of how to induce CNV, including laser induction, lesion excision, processing and different approaches to quantify neoformed vasculature.

Obstetric fistula in Burundi: a comprehensive approach to managing women with this neglected disease.

BACKGROUND: In Burundi, the annual incidence of obstetric fistula is estimated to be 0.2-0.5% of all deliveries, with 1000-2000 new cases per year. Despite this relatively high incidence, national capacity for identifying and managing obstetric fistula is very limited. Thus, in July 2010, Medecins Sans Frontieres (MSF) set up a specialised Obstetric Fistula Centre in Gitega (Gitega Fistula Centre, GFC), the only permanent referral centre for obstetric fistula in Burundi. A comprehensive model of care is offered including psychosocial support, conservative and surgical management, post-operative care and follow-up. We describe this model of care, patient outcomes and the operational challenges. METHODS: Descriptive study using routine programme data. RESULTS: Between July 2010 and December 2011, 470 women with obstetric fistula presented for the first time at GFC, of whom 458 (98%) received treatment. Early urinary catheterization (conservative management) was successful in four out of 35 (11%) women. Of 454 (99%) women requiring surgical management, 394 (87%) were discharged with a closed fistula, of whom 301 (76%) were continent of urine and/or faeces, while 93 (24%) remained incontinent of urine and/or faeces. In 59 (13%) cases, the fistula was complex and could not be closed. Outcome status was unknown for one woman. Median duration of stay at GFC was 39 days (Interquartile range IQR, 31-51 days). CONCLUSION: In a rural African setting, it is feasible to implement a comprehensive package of fistula care using a dedicated fistula facility, and satisfactory surgical repair outcomes can be achieved. Several operational challenges are discussed.

Sunitinib inhibits inflammatory corneal lymphangiogenesis.

PURPOSE: To evaluate the antilymphangiogenic potential of multi-target tyrosine kinase inhibitor sunitinib in corneal neovascularization (NV). METHODS: Inflammatory corneal NV was induced by thermal cauterization applied in the central cornea of mice, to which sunitinib malate was daily administered by gavage or not. At days 6, 11, or 17 post cauterization, lymphatic and blood vessels, as well as inflammatory cells were immunostained and quantified in whole-mounted corneas. RT-PCRs were performed to evidence VEGF-A, VEGF-C, VEGF-D, placental growth factor (PlGF), and soluble vascular endothelial growth factor receptor (VEGFR)-1 and -2 (sVEGFR-1, sVEGFR-2) expressions. Macrophages were isolated from mice peritoneal cavity following thioglycollate injection to produce conditioned medium. The effects of sunitinib were evaluated in vitro in the aortic and lymphatic ring assays in the presence or not of macrophage conditioned medium. RESULTS: Sunitinib treatment drastically reduced pathologic corneal lymphangiogenesis and angiogenesis. Reduced F4/80+ cell infiltration was evidenced in sunitinib-treated mice and was associated to decreased VEGF-A (by 50%, P < 0.01) and VEGF-C (by 35%, P < 0.01) expressions, while VEGF-D and sVEGFR-2 expressions were not affected. In vitro, sunitinib dose-dependently inhibited aortic ring outgrowth, but failed to affect lymphangiogenesis in the lymphatic ring assay. However, macrophage conditioned medium-enhanced angiogenesis and lymphangiogenesis were both strongly counteracted by sunitinib treatment. Mechanistically, sunitinib blocked VEGFR-2 phosphorylation induced by VEGF-A released by macrophages. CONCLUSIONS: Sunitinib exerts antihemangiogenic and antilymphangiogenic effects in vivo by reducing F4/80+ cell recruitment and interacting with their released factors.

MicroRNA-146a is a therapeutic target and biomarker for peripartum cardiomyopathy.

Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR-146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR-146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.

Matrix metalloproteinase-2 governs lymphatic vessel formation as an interstitial collagenase.

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.