segmenting-growth-endothelial-cells-6-well-plates-on-an-orbital

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Agency for Science, Technology and Research (A*STAR)

Nanyang Technological University Singapore

The Academia

Heart failure is a life-threatening condition that carries a considerable emotional and socio-economic burden. As a result of the global increase in the ageing population, sedentary life-style, increased prevalence of risk factors, and improved survival from cardiovascular events, the incidence of heart failure will continue to rise. Despite the advances in current cardiovascular therapies, many patients are not suitable for or may not benefit from conventional treatments. Thus, more effective therapies are required. Transforming growth factor (TGF) β family of cytokines is involved in heart development and dys-regulated TGFβ signalling is commonly associated with fibrosis, aberrant angiogenesis and accelerated progression into heart failure. Therefore, a potential therapeutic pathway is to modulate TGFβ signalling; however, broad blockage of TGFβ signalling may cause unwanted side effects due to its pivotal role in tissue homeostasis. We found that leucine-rich α-2 glycoprotein 1 (LRG1) promotes blood vessel formation via regulating the context-dependent endothelial TGFβ signalling. This review will focus on the interaction between LRG1 and TGFβ signalling, their involvement in the pathogenesis of heart failure, and the potential for LRG1 to function as a novel therapeutic target.

The role of TGFβ1 and LRG1 in cardiac remodelling and heart failure.

Peroxisome proliferator⁻activated receptor (PPAR) β/δ, a ligand-activated transcription factor, is involved in diverse biological processes including cell proliferation, cell differentiation, inflammation and energy homeostasis. Besides its well-established roles in metabolic disorders, PPARβ/δ has been linked to carcinogenesis and was reported to inhibit melanoma cell proliferation, anchorage-dependent clonogenicity and ectopic xenograft tumorigenicity. However, PPARβ/δ's role in tumour progression and metastasis remains controversial.

The Role of PPARβ/δ in Melanoma Metastasis.

Eye diseases and injuries impose a significant clinical problem worldwide. Safe and effective ocular drug delivery is, however, challenging due to the presence of ocular barriers. Here we report a strategy using an eye patch equipped with an array of detachable microneedles. These microneedles can penetrate the ocular surface tissue, and serve as implanted micro-reservoirs for controlled drug delivery. The biphasic drug release kinetics enabled by the double-layered micro-reservoirs largely enhances therapeutic efficacy. Using corneal neovascularization as the disease model, we show that delivery of an anti-angiogenic monoclonal antibody (DC101) by such eye patch produces ~90% reduction of neovascular area. Furthermore, quick release of an anti-inflammatory compound (diclofenac) followed by a sustained release of DC101 provides synergistic therapeutic outcome. The eye patch application is easy and minimally invasive to ensure good patient compliance. Such intraocular drug delivery strategy promises effective home-based treatment of many eye diseases.

Self-implantable double-layered micro-drug-reservoirs for efficient and controlled ocular drug delivery.

The development of new blood vessels from pre-existing vasculature is called angiogenesis. The growth of tumors depends on a network of supplying vessels that provide them with oxygen and nutrients. Pro-angiogenic factors that are secreted by tumors will trigger the sprouting of nearby existing blood vessels towards themselves and therefore researchers have developed targeted therapy towards these pro-angiogenic proteins to inhibit angiogenesis. However, certain pro-angiogenic proteins tend to bypass the inhibition. Thus, instead of targeting these expressed proteins, research towards angiogenesis inhibition had been focused on a deeper scale, epigenetic modifications. Epigenetic regulatory mechanisms are a heritable change in a sequence of stable but reversible gene function modification yet do not affect the DNA primary sequence directly. Methylation of DNA, modification of histone and silencing of micro-RNA (miRNA)-associated gene are currently considered to initiate and sustain epigenetic changes. Recent findings on the subject matter have provided an insight into the mechanism of epigenetic modifications, thus this review aims to present an update on the latest studies.

Epigenetics: The master control of endothelial cell fate in cancer.

Endothelial cell (EC) recruitment is central to the vascularization of tumors. Although several proteoglycans have been implicated in cancer and angiogenesis, their roles in EC recruitment and vascularization during tumorigenesis remain poorly understood. Here, we reveal that Agrin, which is secreted in liver cancer, promotes angiogenesis by recruiting ECs within tumors and metastatic lesions and facilitates adhesion of cancer cells to ECs. In ECs, Agrin-induced angiogenesis and adherence to cancer cells are mediated by Integrin-β1, Lrp4-MuSK pathways involving focal adhesion kinase. Mechanistically, we uncover that Agrin regulates VEGFR2 levels that sustain the angiogenic property of ECs and adherence to cancer cells. Agrin attributes an ECM stiffness-based stabilization of VEGFR2 by enhancing interactions with Integrin-β1-Lrp4 and additionally stimulates endothelial nitric-oxide synthase (e-NOS) signaling. Therefore, we propose that cross-talk between Agrin-expressing cancer and ECs favor angiogenesis by sustaining the VEGFR2 pathway.

A Role of Agrin in Maintaining the Stability of Vascular Endothelial Growth Factor Receptor-2 during Tumor Angiogenesis.

Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated in a timely fashion. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in normal and diabetic wound healing. First, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow-derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, reepithelialization, and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGFβ type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation.

Article	Year
The role of TGFβ1 and LRG1 in cardiac remodelling and heart failure. Biophysical reviews\| PubMed ID: 28509980	2015
The Role of PPARβ/δ in Melanoma Metastasis. International journal of molecular sciences\| PubMed ID: 30241392	2018
Self-implantable double-layered micro-drug-reservoirs for efficient and controlled ocular drug delivery. Nature communications\| PubMed ID: 30401883	2018
Epigenetics: The master control of endothelial cell fate in cancer. Life sciences\| PubMed ID: 31302197	2019
A Role of Agrin in Maintaining the Stability of Vascular Endothelial Growth Factor Receptor-2 during Tumor Angiogenesis. Cell reports\| PubMed ID: 31340156	2019
A Multifunctional Role of Leucine-Rich α-2-Glycoprotein 1 in Cutaneous Wound Healing Under Normal and Diabetic Conditions. Diabetes\| PubMed ID: 32887674	2020

Xiaomeng Wang

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