Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature.

Dysfunctional pericytes and disruption of adherens or tight junctions are related to many microvascular diseases, including diabetic retinopathy. In this context, visualizing retinal vascular architecture becomes essential for understanding retinal vascular disease pathophysiology. Although flat mounts provide a demonstration of the retinal blood vasculature, they often lack a clear view of microaneurysms and capillary architecture. Trypsin and elastase digestion are the two techniques for isolating retinal vasculatures in rats, mice, and other animal models. Our observations in the present study reveal that trypsin digestion impacts the association between pericytes and endothelial cells. In contrast, elastase digestion effectively preserves these features in the blood vessels. Furthermore, trypsin digestion disrupts endothelial adherens and tight junctions that elastase digestion does not. Therefore, elastase digestion emerges as a superior technique for isolating retinal vessels, which can be utilized to collect reliable and consistent data to comprehend the pathophysiology of disorders involving microvascular structures.

Long Non-Coding RNAs and Proliferative Retinal Diseases.

Retinopathy refers to disorders that affect the retina of the eye, which are frequently caused by damage to the retina's vascular system. This causes leakage, proliferation, or overgrowth of blood vessels through the retina, which can lead to retinal detachment or breakdown, resulting in vision loss and, in rare cases, blindness. In recent years, high-throughput sequencing has significantly hastened the discovery of new long non-coding RNAs (lncRNAs) and their biological functions. LncRNAs are rapidly becoming recognized as critical regulators of several key biological processes. Current breakthroughs in bioinformatics have resulted in the identification of several lncRNAs that may have a role in retinal disorders. Nevertheless, mechanistic investigations have yet to reveal the relevance of these lncRNAs in retinal disorders. Using lncRNA transcripts for diagnostic and/or therapeutic purposes may aid in the development of appropriate treatment regimens and long-term benefits for patients, as traditional medicines and antibody therapy only provide temporary benefits that must be repeated. In contrast, gene-based therapies can provide tailored, long-term treatment solutions. Here, we will discuss how different lncRNAs affect different retinopathies, including age-related macular degeneration (AMD), diabetic retinopathy (DR), central retinal vein occlusion (CRVO), proliferative vitreoretinopathy (PVR), and retinopathy of prematurity (ROP), which can cause visual impairment and blindness, and how these retinopathies can be identified and treated using lncRNAs.

Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization.

Vascular adhesion molecules play an important role in various immunological disorders, particularly in cancers. However, little is known regarding the role of these adhesion molecules in proliferative retinopathies. We observed that IL-33 regulates VCAM-1 expression in human retinal endothelial cells and that genetic deletion of IL-33 reduces hypoxia-induced VCAM-1 expression and retinal neovascularization in C57BL/6 mice. We found that VCAM-1 via JunB regulates IL-8 promoter activity and expression in human retinal endothelial cells. In addition, our study outlines the regulatory role of VCAM-1-JunB-IL-8 signaling on retinal endothelial cell sprouting and angiogenesis. Our RNA sequencing results show an induced expression of CXCL1 (a murine functional homolog of IL-8) in the hypoxic retina, and intravitreal injection of VCAM-1 siRNA not only decreases hypoxia-induced VCAM-1-JunB-CXCL1 signaling but also reduces OIR-induced sprouting and retinal neovascularization. These findings suggest that VCAM-1-JunB-IL-8 signaling plays a crucial role in retinal neovascularization, and its antagonism might provide an advanced treatment option for proliferative retinopathies.

New-age vaccine adjuvants, their development, and future perspective.

In the present scenario, immunization is of utmost importance as it keeps us safe and protects us from infectious agents. Despite the great success in the field of vaccinology, there is a need to not only develop safe and ideal vaccines to fight deadly infections but also improve the quality of existing vaccines in terms of partial or inconsistent protection. Generally, subunit vaccines are known to be safe in nature, but they are mostly found to be incapable of generating the optimum immune response. Hence, there is a great possibility of improving the potential of a vaccine in formulation with novel adjuvants, which can effectively impart superior immunity. The vaccine(s) in formulation with novel adjuvants may also be helpful in fighting pathogens of high antigenic diversity. However, due to the limitations of safety and toxicity, very few human-compatible adjuvants have been approved. In this review, we mainly focus on the need for new and improved vaccines; the definition of and the need for adjuvants; the characteristics and mechanisms of human-compatible adjuvants; the current status of vaccine adjuvants, mucosal vaccine adjuvants, and adjuvants in clinical development; and future directions.

IL-33 via PKCµ/PRKD1 Mediated α-Catenin Phosphorylation Regulates Endothelial Cell-Barrier Integrity and Ischemia-Induced Vascular Leakage.

Angiogenesis, neovascularization, and vascular remodeling are highly dynamic processes, where endothelial cell-cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity, and barrier function. The cadherin-catenin adhesion complex is a key contributor to inner blood-retinal barrier (iBRB) integrity and dynamic cell movements. However, the pre-eminent role of cadherins and their associated catenins in iBRB structure and function is not fully understood. Using a murine model of oxygen-induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we try to understand the significance of IL-33 on retinal endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability. Using electric cell-substrate impedance sensing (ECIS) analysis and FITC-dextran permeability assay, we observed that IL-33 at a 20 ng/mL concentration induced endothelial-barrier disruption in HRMVECs. The adherens junction (AJs) proteins play a prominent role in the selective diffusion of molecules from the blood to the retina and in maintaining retinal homeostasis. Therefore, we looked for the involvement of adherens junction proteins in IL-33-mediated endothelial dysfunction. We observed that IL-33 induces α-catenin phosphorylation at serine/threonine (Ser/Thr) residues in HRMVECs. Furthermore, mass-spectroscopy (MS) analysis revealed that IL-33 induces the phosphorylation of α-catenin at Thr654 residue in HRMVECs. We also observed that PKCµ/PRKD1-p38 MAPK signaling regulates IL-33-induced α-catenin phosphorylation and retinal endothelial cell-barrier integrity. Our OIR studies revealed that genetic deletion of IL-33 resulted in reduced vascular leakage in the hypoxic retina. We also observed that the genetic deletion of IL-33 reduced OIR-induced PKCµ/PRKD1-p38 MAPK-α-catenin signaling in the hypoxic retina. Therefore, we conclude that IL-33-induced PKCµ/PRKD1-p38 MAPK-α-catenin signaling plays a significant role in endothelial permeability and iBRB integrity.

Role of G-Proteins and GPCRs in Cardiovascular Pathologies.

Cell signaling is a fundamental process that enables cells to survive under various ecological and environmental contexts and imparts tolerance towards stressful conditions. The basic machinery for cell signaling includes a receptor molecule that senses and receives the signal. The primary form of the signal might be a hormone, light, an antigen, an odorant, a neurotransmitter, etc. Similarly, heterotrimeric G-proteins principally provide communication from the plasma membrane G-protein-coupled receptors (GPCRs) to the inner compartments of the cells to control various biochemical activities. G-protein-coupled signaling regulates different physiological functions in the targeted cell types. This review article discusses G-proteins' signaling and regulation functions and their physiological relevance. In addition, we also elaborate on the role of G-proteins in several cardiovascular diseases, such as myocardial ischemia, hypertension, atherosclerosis, restenosis, stroke, and peripheral artery disease.

The Biochemistry and Physiology of A Disintegrin and Metalloproteinases (ADAMs and ADAM-TSs) in Human Pathologies.

Metalloproteinases are a group of proteinases that plays a substantial role in extracellular matrix remodeling and its molecular signaling. Among these metalloproteinases, ADAMs (a disintegrin and metalloproteinases) and ADAM-TSs (ADAMs with thrombospondin domains) have emerged as highly efficient contributors mediating proteolytic processing of various signaling molecules. ADAMs are transmembrane metalloenzymes that facilitate the extracellular domain shedding of membrane-anchored proteins, cytokines, growth factors, ligands, and their receptors and therefore modulate their biological functions. ADAM-TSs are secretory, and soluble extracellular proteinases that mediate the cleavage of non-fibrillar extracellular matrix proteins. ADAMs and ADAM-TSs possess pro-domain, metalloproteinase, disintegrin, and cysteine-rich domains in common, but ADAM-TSs have characteristic thrombospondin motifs instead of the transmembrane domain. Most ADAMs and ADAM-TSs are activated by cleavage of pro-domain via pro-protein convertases at their N-terminus, hence directing them to various signaling pathways. In this article, we are discussing not only the structure and regulation of ADAMs and ADAM-TSs, but also the importance of these metalloproteinases in various human pathophysiological conditions like cardiovascular diseases, colorectal cancer, autoinflammatory diseases (sepsis/rheumatoid arthritis), Alzheimer's disease, proliferative retinopathies, and infectious diseases. Therefore, based on the emerging role of ADAMs and ADAM-TSs in various human pathologies, as summarized in this review, these metalloproteases can be considered as critical therapeutic targets and diagnostic biomarkers.

Inflammation and retinal degenerative diseases.

Vision is an ability that depends on the precise structure and functioning of the retina. Any kind of stress or injury can disrupt the retinal architecture and leads to vision impairment, vision loss, and blindness. Immune system and immune response function maintain homeostasis in the microenvironment. Several genetic, metabolic, and environmental factors may alter retinal homeostasis, and these events may initiate various inflammatory cascades. The prolonged inflammatory state may contribute to the initiation and development of retinal disorders such as glaucoma, age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa, which pose a threat to vision. In the current review, we attempted to provide sufficient evidence on the role of inflammation in these retinal disorders. Moreover, this review paves the way to focus on therapeutic targets of the disease, which are found to be promising.

Nanotechnology for colorectal cancer detection and treatment.

Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer-related mortality in the United States. Across the globe, people in the age group older than 50 are at a higher risk of CRC. Genetic and environmental risk factors play a significant role in the development of CRC. If detected early, CRC is preventable and treatable. Currently, available screening methods and therapies for CRC treatment reduce the incidence rate among the population, but the micrometastasis of cancer may lead to recurrence. Therefore, the challenge is to develop an alternative therapy to overcome this complication. Nanotechnology plays a vital role in cancer treatment and offers targeted chemotherapies directly and selectively to cancer cells, with enhanced therapeutic efficacy. Additionally, nanotechnology elevates the chances of patient survival in comparison to traditional chemotherapies. The potential of nanoparticles includes that they may be used simultaneously for diagnosis and treatment. These exciting properties of nanoparticles have enticed researchers worldwide to unveil their use in early CRC detection and as effective treatment. This review discusses contemporary methods of CRC screening and therapies for CRC treatment, while the primary focus is on the theranostic approach of nanotechnology in CRC treatment and its prospects. In addition, this review aims to provide knowledge on the advancement of nanotechnology in CRC and as a starting point for researchers to think about new therapeutic approaches using nanotechnology.

IL-33 enhances Jagged1 mediated NOTCH1 intracellular domain (NICD) deubiquitination and pathological angiogenesis in proliferative retinopathy.

Pathological retinal neovascularization (NV) is a clinical manifestation of various proliferative retinopathies, and treatment of NV using anti-VEGF therapies is not selective, as it also impairs normal retinal vascular growth and function. Here, we show that genetic deletion or siRNA-mediated downregulation of IL-33 reduces pathological NV in a murine model of oxygen-induced retinopathy (OIR) with no effect on the normal retinal repair. Furthermore, our fluorescent activated cell sorting (FACS) data reveals that the increase in IL-33 expression is in endothelial cells (ECs) of the hypoxic retina and conditional genetic deletion of IL-33 in retinal ECs reduces pathological NV. In vitro studies using human retinal microvascular endothelial cells (HRMVECs) show that IL-33 induces sprouting angiogenesis and requires NFkappaB-mediated Jagged1 expression and Notch1 activation. Our data also suggest that IL-33 enhances de-ubiquitination and stabilization of Notch1 intracellular domain via its interaction with BRCA1-associated protein 1 (BAP1) and Numb in HRMVECs and a murine model of OIR.

Nano-Biomaterials for Retinal Regeneration.

Nanoscience and nanotechnology have revolutionized key areas of environmental sciences, including biological and physical sciences. Nanoscience is useful in interconnecting these sciences to find new hybrid avenues targeted at improving daily life. Pharmaceuticals, regenerative medicine, and stem cell research are among the prominent segments of biological sciences that will be improved by nanostructure innovations. The present review was written to present a comprehensive insight into various emerging nanomaterials, such as nanoparticles, nanowires, hybrid nanostructures, and nanoscaffolds, that have been useful in mice for ocular tissue engineering and regeneration. Furthermore, the current status, future perspectives, and challenges of nanotechnology in tracking cells or nanostructures in the eye and their use in modified regenerative ophthalmology mechanisms have also been proposed and discussed in detail. In the present review, various research findings on the use of nano-biomaterials in retinal regeneration and retinal remediation are presented, and these findings might be useful for future clinical applications.

The Role of Inflammation in Retinal Neurodegeneration and Degenerative Diseases.

Retinal neurodegeneration is predominantly reported as the apoptosis or impaired function of the photoreceptors. Retinal degeneration is a major causative factor of irreversible vision loss leading to blindness. In recent years, retinal degenerative diseases have been investigated and many genes and genetic defects have been elucidated by many of the causative factors. An enormous amount of research has been performed to determine the pathogenesis of retinal degenerative conditions and to formulate the treatment modalities that are the critical requirements in this current scenario. Encouraging results have been obtained using gene therapy. We provide a narrative review of the various studies performed to date on the role of inflammation in human retinal degenerative diseases such as age-related macular degeneration, inherited retinal dystrophies, retinitis pigmentosa, Stargardt macular dystrophy, and Leber congenital amaurosis. In addition, we have highlighted the pivotal role of various inflammatory mechanisms in the progress of retinal degeneration. This review also offers an assessment of various therapeutic approaches, including gene-therapies and stem-cell-based therapies, for degenerative retinal diseases.

Thrombin-Par1 signaling axis disrupts COP9 signalosome subunit 3-mediated ABCA1 stabilization in inducing foam cell formation and atherogenesis.

ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) play a vital role in promoting cholesterol efflux. Although, the dysregulation of these transporters was attributed as one of the mechanisms of atherogenesis, what renders their dysfunction is not well explored. Previously, we have reported that thrombin without having any effect on ABCG1 levels depletes ABCA1 levels affecting cholesterol efflux. In this study, we explored the mechanisms underlying thrombin-induced depletion of ABCA1 levels both in macrophages and smooth muscle cells. Under normal physiological conditions, COP9 signalosome subunit 3 (CSN3) was found to exist in complex with ABCA1 and in the presence of proatherogenic stimulants such as thrombin, ABCA1 was phosphorylated and dissociated from CSN3, leading to its degradation. Forced expression of CSN3 inhibited thrombin-induced ABCA1 ubiquitination and degradation, restored cholesterol efflux and suppressed foam cell formation. In Western diet (WD)-fed ApoE-/- mice, CSN3 was also disassociated from ABCA1 otherwise remained as a complex in Chow diet (CD)-fed ApoE-/- mice. Interestingly, depletion of CSN3 levels in WD-fed ApoE-/- mice significantly lowered ABCA1 levels, inhibited cholesterol efflux and intensified foam cell formation exacerbating the lipid laden atherosclerotic plaque formation. Mechanistic studies have revealed the involvement of Par1-Gα12-Pyk2-Gab1-PKCθ signaling in triggering phosphorylation of ABCA1 and its disassociation from CSN3 curtailing cholesterol efflux and amplifying foam cell formation. In addition, although both CSN3 and ABCA1 were found to be colocalized in human non-lesion coronary arteries, their levels were decreased as well as dissociated from each other in advanced atherosclerotic lesions. Together, these observations reveal for the first time an anti-atherogenic role of CSN3 and hence, designing therapeutic drugs protecting its interactions with ABCA1 could be beneficial against atherosclerosis.

PKCθ-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization.

Pathological retinal neovascularization is the most common cause of vision loss. PKCθ has been shown to play a role in type 2 diabetes, which is linked to retinal neovascularization. Based on these clues, we have studied the role of PKCθ and its downstream target genes JunB and VEGFR3 in retinal neovascularization using global and tissue-specific knockout mouse models along with molecular biological approaches. Here, we show that vascular endothelial growth factor A (VEGFA) induces PKCθ phosphorylation in human retinal microvascular endothelial cells (HRMVECs) and downregulation of its levels attenuates VEGFA-induced HRMVECs migration, sprouting and tube formation. Furthermore, the whole body deletion of PKCθ or EC-specific deletion of its target gene JunB inhibited hypoxia-induced retinal EC proliferation, tip cell formation and neovascularization. VEGFA also induced VEGFR3 expression via JunB downstream to PKCθ in the regulation of HRMVEC migration, sprouting, and tube formation in vitro and OIR-induced retinal EC proliferation, tip cell formation and neovascularization in vivo. In addition, VEGFA-induced VEGFR3 expression requires VEGFR2 activation upstream to PKCθ-JunB axis both in vitro and in vivo. Depletion of VEGFR2 or VEGFR3 levels attenuated VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and retinal neovascularization in vivo and it appears that these events were dependent on STAT3 activation. Furthermore, the observations using soluble VEGFR3 indicate that VEGFR3 mediates its effects on retinal neovascularization in a ligand dependent and independent manner downstream to VEGFR2. Together, these observations suggest that PKCθ-dependent JunB-mediated VEGFR3 expression targeting STAT3 activation is required for VEGFA/VEGFR2-induced retinal neovascularization.

NFATc1-E2F1-LMCD1-Mediated IL-33 Expression by Thrombin Is Required for Injury-Induced Neointima Formation.

Objective- IL (interleukin)-33 has been shown to play a role in endothelial dysfunction, but its role in atherosclerosis is controversial. Therefore, the purpose of this study is to examine its role in vascular wall remodeling following injury. Approach and Results- Thrombin induced IL-33 expression in a time-dependent manner in human aortic smooth muscle cells and inhibition of its activity by its neutralizing antibody suppressed thrombin induced human aortic smooth muscle cell migration but not DNA synthesis. In exploring the mechanisms, we found that Par1 (protease-activated receptor 1), Gαq/11 (Gα protein q/11), PLCß3 (phospholipase Cß3), NFATc1 (nuclear factor of activated T cells), E2F1 (E2F transcription factor 1), and LMCD1 (LIM and cysteine-rich domains protein 1) are involved in thrombin-induced IL-33 expression and migration. Furthermore, we identified an NFAT-binding site at -100 nt that mediates thrombin-induced IL-33 promoter activity. Interestingly, we observed that NFATc1, E2F1, and LMCD1 bind to NFAT site in response to thrombin and found that LMCD1, while alone has no significant effect, enhanced either NFATc1 or E2F1-dependent IL-33 promoter activity. In addition, we found that guidewire injury induces IL-33 expression in SMC and its neutralizing antibodies substantially reduce SMC migration and neointimal growth in vivo. Increased expression of IL-33 was also observed in human atherosclerotic lesions as compared to arteries without any lesions. Conclusions- The above findings reveal for the first time that thrombin-induced human aortic smooth muscle cell migration and injury-induced neointimal growth require IL-33 expression. In addition, thrombin-induced IL-33 expression requires LMCD1 enhanced combinatorial activation of NFATc1 and E2F1.