Lymphatic Connexins and Pannexins in Health and Disease

This review highlights current knowledge on the expression and function of connexins and pannexins, transmembrane channel proteins that play an important role in intercellular communication, in both the developing and mature lymphatic vasculature. A particular focus is given to the involvement of these proteins in functions of the healthy lymphatic system. We describe their influence on the maintenance of extracellular fluid homeostasis, immune cell trafficking to draining lymph nodes and dietary nutrient absorption by intestinal villi. Moreover, new insights into connexin mutations in primary and secondary lymphedema as well as on the implication of lymphatic connexins and pannexins in acquired cardiovascular diseases are discussed, allowing for a better understanding of the role of these proteins in pathologies linked to dysfunctions in the lymphatic system.     

Regulation of VEGFR Signalling in Lymphatic Vascular Development and Disease: An Update

The importance of lymphatic vessels in a myriad of human diseases is rapidly gaining recognition; lymphatic vessel dysfunction is a feature of disorders including congenital lymphatic anomalies, primary lymphoedema and obesity, while improved lymphatic vessel function increases the efficacy of immunotherapy for cancer and neurological disease and promotes cardiac repair following myocardial infarction. Understanding how the growth and function of lymphatic vessels is precisely regulated therefore stands to inform the development of novel therapeutics applicable to a wide range of human diseases. Lymphatic vascular development is initiated during embryogenesis following establishment of the major blood vessels and the onset of blood flow. Lymphatic endothelial progenitor cells arise from a combination of venous and non-venous sources to generate the initial lymphatic vascular structures in the vertebrate embryo, which are then further ramified and remodelled to elaborate an extensive lymphatic vascular network. Signalling mediated via vascular endothelial growth factor (VEGF) family members and vascular endothelial growth factor receptor (VEGFR) tyrosine kinases is crucial for development of both the blood and lymphatic vascular networks, though distinct components are utilised to different degrees in each vascular compartment. Although much is known about the regulation of VEGFA/VEGFR2 signalling in the blood vasculature, less is understood regarding the mechanisms by which VEGFC/VEGFD/VEGFR3 signalling is regulated during lymphatic vascular development. This review will focus on recent advances in our understanding of the cellular and molecular mechanisms regulating VEGFA-, VEGFC- and VEGFD-mediated signalling via VEGFRs which are important for driving the construction of lymphatic vessels during development and disease.     

The Kinetics of Lymphatic Dysfunction and Leukocyte Expansion in the Draining Lymph Node during LTB4 Antagonism in a Mouse Model of Lymphedema

The mechanisms of lymphedema development are not well understood, but emerging evidence highlights the crucial role the immune system plays in driving its progression. It is well known that lymphatic function deteriorates as lymphedema progresses; however, the connection between this progressive loss of function and the immune-driven changes that characterize the disease has not been well established. In this study, we assess changes in leukocyte populations in lymph nodes within the lymphatic drainage basin of the tissue injury site (draining lymph nodes, dLNs) using a mouse tail model of lymphedema in which a pair of draining collecting vessels are left intact. We additionally quantify lymphatic pump function using established near infrared (NIR) lymphatic imaging methods and lymph-draining nanoparticles (NPs) synthesized and employed by our team for lymphatic tissue drug delivery applications to measure lymphatic transport to and resulting NP accumulation within dLNs associated with swelling following surgery. When applied to assess the effects of the anti-inflammatory drug bestatin, which has been previously shown to be a possible treatment for lymphedema, we find lymph-draining NP accumulation within dLNs and lymphatic function to increase as lymphedema progresses, but no significant effect on leukocyte populations in dLNs or tail swelling. These results suggest that ameliorating this loss of lymphatic function is not sufficient to reverse swelling in this surgically induced disease model that better recapitulates the extent of lymphatic injury seen in human lymphedema. It also suggests that loss of lymphatic function during lymphedema may be driven by immune-mediated mechanisms coordinated in dLNs. Our work indicates that addressing both lymphatic vessel dysfunction and immune cell expansion within dLNs may be required to prevent or reverse lymphedema when partial lymphatic function is sustained.     

Decreased Lymphangiogenic Activities and Genes Expression of Cord Blood Lymphatic Endothelial Progenitor Cells (VEGFR3+/Pod+/CD11b+ Cells) in Patient with Preeclampsia

The abnormal development or disruption of the lymphatic vasculature has been implicated in metabolic and hypertensive diseases. Recent evidence suggests that the offspring exposed to preeclampsia (PE) in utero are at higher risk of long-term health problems, such as cardiovascular and metabolic diseases in adulthood, owing to in utero fetal programming. We aimed to investigate lymphangiogenic activities in the lymphatic endothelial progenitor cells (LEPCs) of the offspring of PE. Human umbilical cord blood LEPCs from pregnant women with severe PE (n = 10) and gestationally matched normal pregnancies (n = 10) were purified with anti-vascular endothelial growth factor receptor 3 (VEGFR3)/podoplanin/CD11b microbeads using a magnetic cell sorter device. LEPCs from PE displayed significantly delayed differentiation and reduced formation of lymphatic endothelial cell (LEC) colonies compared with the LEPCs from normal pregnancies. LECs differentiated from PE-derived LEPCs exhibited decreased tube formation, migration, proliferation, adhesion, wound healing, and 3D-sprouting activities as well as increased lymphatic permeability through the disorganization of VE-cadherin junctions, compared with the normal pregnancy-derived LECs. In vivo, LEPCs from PE showed significantly reduced lymphatic vessel formation compared to the LEPCs of the normal pregnancy. Gene expression analysis revealed that compared to the normal pregnancy-derived LECs, the PE-derived LECs showed a significant decrease in the expression of pro-lymphangiogenic genes (GREM1, EPHB3, VEGFA, AMOT, THSD7A, ANGPTL4, SEMA5A, FGF2, and GBX2). Collectively, our findings demonstrate, for the first time, that LEPCs from PE have reduced lymphangiogenic activities in vitro and in vivo and show the decreased expression of pro-lymphangiogenic genes. This study opens a new avenue for investigation of the molecular mechanism of LEPC differentiation and lymphangiogenesis in the offspring of PE and subsequently may impact the treatment of long-term health problems such as cardiovascular and metabolic disorders of offspring with abnormal development of lymphatic vasculature.     

Stretch-Induced Tenomodulin Expression Promotes Tenocyte Migration via F-Actin and Chromatin Remodeling

The mechanosensitive gene tenomodulin (Tnmd) is implicated in tendon maturation and repair. However, the mechanism by which mechanical loading regulates Tnmd’s expression and its role in tenocyte migration is yet to be defined. Here, we show that Tnmd and migration were upregulated in uniaxial cyclic stress-stimulated tenocytes. The knockdown of Tnmd reduced cell migration in the presence and absence of mechanical loading, suggesting that Tnmd is involved in tenocyte migration. Moreover, the treatment of stress-stimulated tenocytes with the actin inhibitor latrunculin (Lat A), histone acetyltransferase inhibitor anacardic acid (ANA), or histone demethylases inhibitor GSK-J4 suppressed Tnmd expression and tenocyte migration. These results show that actin stress fiber formation and chromatin decondensation regulates Tnmd expression, which might then regulate tenocyte migration. Thus, this study proposes the involvement of the actin and chromatin mechanotransduction pathway in the regulation of Tnmd and reveals a novel role of Tnmd in tenocyte migration. The identification of Tnmd function in tenocyte migration provides insight into the molecular mechanisms involved in Tnmd-mediated tendon repair.     

Involvement of the FAK Network in Pathologies Related to Altered Mechanotransduction

Mechanotransduction is a physiological process in which external mechanical stimulations are perceived, interpreted, and translated by cells into biochemical signals. Mechanical stimulations exerted by extracellular matrix stiffness and cell–cell contacts are continuously applied to living cells, thus representing a key pivotal trigger for cell homeostasis, survival, and function, as well as an essential factor for proper organ development and metabolism. Indeed, a deregulation of the mechanotransduction process consequent to gene mutations or altered functions of proteins involved in perceiving cellular and extracellular mechanics can lead to a broad range of diseases, from muscular dystrophies and cardiomyopathies to cancer development and metastatization. Here, we recapitulate the involvement of focal adhesion kinase (FAK) in the cellular conditions deriving from altered mechanotransduction processes.     

Protein Folding and Mechanisms of Proteostasis

Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis.     

Epigenetic Regulation of Autophagy in Cardiovascular Pathobiology

Cardiovascular diseases (CVDs) are the number one cause of debilitation and mortality worldwide, with a need for cost-effective therapeutics. Autophagy is a highly conserved catabolic recycling pathway triggered by various intra- or extracellular stimuli to play an essential role in development and pathologies, including CVDs. Accordingly, there is great interest in identifying mechanisms that govern autophagic regulation. Autophagic regulation is very complex and multifactorial that includes epigenetic pathways, such as histone modifications to regulate autophagy-related gene expression, decapping-associated mRNA degradation, microRNAs, and long non-coding RNAs; pathways are also known to play roles in CVDs. Molecular understanding of epigenetic-based pathways involved in autophagy and CVDs not only will enhance the understanding of CVDs, but may also provide novel therapeutic targets and biomarkers for CVDs.     

Transmembrane TNF and Its Receptors TNFR1 and TNFR2 in Mycobacterial Infections

Tumor necrosis factor (TNF) is one of the main cytokines regulating a pro-inflammatory environment. It has been related to several cell functions, for instance, phagocytosis, apoptosis, proliferation, mitochondrial dynamic. Moreover, during mycobacterial infections, TNF plays an essential role to maintain granuloma formation. Several effector mechanisms have been implicated according to the interactions of the two active forms, soluble TNF (solTNF) and transmembrane TNF (tmTNF), with their receptors TNFR1 and TNFR2. We review the impact of these interactions in the context of mycobacterial infections. TNF is tightly regulated by binding to receptors, however, during mycobacterial infections, upstream activation signalling pathways may be influenced by key regulatory factors either at the membrane or cytosol level. Detailing the structure and activation pathways used by TNF and its receptors, such as its interaction with solTNF/TNFRs versus tmTNF/TNFRs, may bring a better understanding of the molecular mechanisms involved in activation pathways which can be helpful for the development of new therapies aimed at being more efficient against mycobacterial infections.     

Human gene control by vital oncogenes: revisiting a theoretical model and its implications for targeted cancer therapy

An important assumption of our current understanding of the mechanisms of carcinogenesis has been the belief that clarification of the cancer process would inevitably reveal some of the crucial mechanisms of normal human gene regulation. Since the momentous work of Bishop and Varmus, both the molecular and the biochemical processes underlying the events in the development of cancer have become increasingly clear. The identification of cellular signaling pathways and the role of protein kinases in the events leading to gene activation have been critical to our understanding not only of normal cellular gene control mechanisms, but also have clarified some of the important molecular and biochemical events occurring within a cancer cell. We now know that oncogenes are dysfunctional proto-oncogenes and that dysfunctional tumor suppressor genes contribute to the cancer process. Furthermore, Weinstein and others have hypothesized the phenomenon of oncogene addiction as a distinct characteristic of the malignant cell. It can be assumed that cancer cells, indeed, become dependent on such vital oncogenes. The products of these vital oncogenes, such as c-myc, may well be the Achilles heel by which targeted molecular therapy may lead to truly personalized cancer therapy. The remaining problem is the need to introduce relevant molecular diagnostic tests such as genome microarray analysis and proteomic methods, especially protein kinase identification arrays, for each individual patient. Genome wide association studies on cancers with gene analysis of single nucleotide and other mutations in functional proto-oncogenes will, hopefully, identify dysfunctional proto-oncogenes and allow the development of more specific targeted drugs directed against the protein products of these vital oncogenes. In 1984 Willis proposed a molecular and biochemical model for eukaryotic gene regulation suggesting how proto-oncogenes might function within the normal cell. That model predicted the existence of vital oncogenes and can now be used to hypothesize the biochemical and molecular mechanisms that drive the processes leading to disruption of the gene regulatory machinery, resulting in the transformation of normal cells into cancer.     

Expression of Lymphatic Markers in the Berger’s Space and Bursa Premacularis

We previously reported that the bursa premacularis (BPM), a peculiar vitreous structure located above the macula, contains numerous cells expressing markers of lymphatic endothelial cells, such as podoplanin and LYVE-1. Herein, we examined the expression of lymphatic markers in the Berger’s space (BS), BPM, and vitreous core (VC). BS, BPM, and VC specimens were selectively collected in macular hole and epiretinal membrane patients during vitrectomy and were then immunostained with antibodies for podoplanin, LYVE-1, and fibrillin-1 and -2. By visualization using triamcinolone acetonide, the BS was recognized as a sac-like structure with a septum located behind the lens as well as BPM. Those tissues adhered to the lens or retina in a circular manner by means of a ligament-like structure. Immunostaining showed intense expression of podoplanin and LYVE-1 in the BS. Both BS and BPM stained strongly positive for fibrillin-1 and -2. The VC was faintly stained with antibodies for those lymph-node markers. Our findings indicate that both BS and BPM possibly belong to the lymphatic system, such as lymph nodes, draining excess fluid and waste products into lymphatic vessels in the dura mater of the optic nerve and the ciliary body, respectively, via intravitreal canals.     

Melanoma Brain Metastasis: Mechanisms,Models, and Medicine

The development of brain metastases in patients with advanced stage melanoma is common, but the molecular mechanisms responsible for their development are poorly understood. Melanoma brain metastases cause significant morbidity and mortality and confer a poor prognosis; traditional therapies including whole brain radiation, stereotactic radiotherapy, or chemotherapy yield only modest increases in overall survival (OS) for these patients. While recently approved therapies have significantly improved OS in melanoma patients, only a small number of studies have investigated their efficacy in patients with brain metastases. Preliminary data suggest that some responses have been observed in intracranial lesions, which has sparked new clinical trials designed to evaluate the efficacy in melanoma patients with brain metastases. Simultaneously, recent advances in our understanding of the mechanisms of melanoma cell dissemination to the brain have revealed novel and potentially therapeutic targets. In this review, we provide an overview of newly discovered mechanisms of melanoma spread to the brain, discuss preclinical models that are being used to further our understanding of this deadly disease and provide an update of the current clinical trials for melanoma patients with brain metastases.     

Expression and Role of Ubiquitin-Specific Peptidases in Osteoblasts

The ubiquitin-proteasome system regulates biological processes in normal and diseased states. Recent investigations have focused on ubiquitin-dependent modifications and their impacts on cellular function, commitment, and differentiation. Ubiquitination is reversed by deubiquitinases, including ubiquitin-specific peptidases (USPs), whose roles have been widely investigated. In this review, we explore recent findings highlighting the regulatory functions of USPs in osteoblasts and providing insight into the molecular mechanisms governing their actions during bone formation. We also give a brief overview of our work on USP53, a target of PTH in osteoblasts and a regulator of mesenchymal cell lineage fate decisions. Emerging evidence addresses questions pertaining to the complex layers of regulation exerted by USPs on osteoblast signaling. We provide a short overview of our and others’ understanding of how USPs modulate osteoblastogenesis. However, further studies using knockout mouse models are needed to fully understand the mechanisms underpinning USPs actions.     

Modeling Neoplastic Growth in Renal Cell Carcinoma and Polycystic Kidney Disease

Renal cell carcinoma (RCC) and autosomal dominant polycystic kidney disease (ADPKD) share several characteristics, including neoplastic cell growth, kidney cysts, and limited therapeutics. As well, both exhibit impaired vasculature and compensatory VEGF activation of angiogenesis. The PI3K/AKT/mTOR and Ras/Raf/ERK pathways play important roles in regulating cystic and tumor cell proliferation and growth. Both RCC and ADPKD result in hypoxia, where HIF-α signaling is activated in response to oxygen deprivation. Primary cilia and altered cell metabolism may play a role in disease progression. Non-coding RNAs may regulate RCC carcinogenesis and ADPKD through their varied effects. Drosophila exhibits remarkable conservation of the pathways involved in RCC and ADPKD. Here, we review the progress towards understanding disease mechanisms, partially overlapping cellular and molecular dysfunctions in RCC and ADPKD and reflect on the potential for the agile Drosophila genetic model to accelerate discovery science, address unresolved mechanistic aspects of these diseases, and perform rapid pharmacological screens.     

Comparative Proteomic Analysis of Developmental Changes in P-Type Cytoplasmic Male Sterile and Maintainer Anthers in Wheat

Cytoplasmic male sterility (CMS) plays an important role in the application of heterosis in wheat (Triticum aestivum L.). However, the molecular mechanism underlying CMS remains unknown. This study provides a comprehensive morphological and proteomic analysis of the anthers of a P-type CMS wheat line (P) and its maintainer line, Yanshi 9 hao (Y). Cytological observations indicated that the P-type CMS line shows binucleate microspore abortion. In this line, the tapetum degraded early, leading to anther cuticle defects, which could not provide the nutrition needed for microspore development in a timely manner, thus preventing the development of the microspore to the normal binucleate stage. Proteomic analysis revealed novel proteins involved in P-type CMS. Up to 2576 differentially expressed proteins (DEPs) were quantified in all anthers, and these proteins were significantly enriched in oxidative phosphorylation, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), starch and sucrose metabolism, phenylpropanoid biosynthesis, and pyruvate metabolism pathways. These proteins may comprise a network that regulates male sterility in wheat. Based on the function analysis of DEPs involved in the complex network, we concluded that the P-type CMS line may be due to cellular dysfunction caused by disturbed carbohydrate metabolism, inadequate energy supply, and disturbed protein synthesis. These results provide insights into the molecular mechanism underlying male sterility and serve as a valuable resource for researchers in plant biology, in general, and plant sexual reproduction, in particular.     

Marine Polysaccharide Networks and Diatoms at the Nanometric Scale

Despite many advances in research on photosynthetic carbon fixation in marine diatoms, the biophysical and biochemical mechanisms of extracellular polysaccharide production remain significant challenges to be resolved at the molecular scale in order to proceed toward an understanding of their functions at the cellular level, as well as their interactions and fate in the ocean. This review covers studies of diatom extracellular polysaccharides using atomic force microscopy (AFM) imaging and the quantification of physical forces. Following a brief summary of the basic principle of the AFM experiment and the first AFM studies of diatom extracellular polymeric substance (EPS), we focus on the detection of supramolecular structures in polysaccharide systems produced by marine diatoms. Extracellular polysaccharide fibrils, attached to the diatom cell wall or released into the surrounding seawater, form distinct supramolecular assemblies best described as gel networks. AFM makes characterization of the diatom polysaccharide networks at the micro and nanometric scales and a clear distinction between the self-assembly and self-organization of these complex systems in marine environments possible.