This review presents a holistic view on the translational potential of the interplay between stromal cells and cancer cells. This interplay is currently being employed for the development of promising preclinical and clinical biomarkers, and the design of small molecule inhibitors, antibodies and small RNAs for (combinatorial) cancer treatment options. In addition, nano-carriers, tissue scaffolds and 3-D based matrices are being developed to precisely and safely deliver these compounds.
Fluorophores are ubiquitous in nature. Naturally occurring fluorophores are exceptionally stable and have high quantum yield. Several natural systems have acquired fluorescent signature due to the presence of these fluorophores. Systematic attempt to harvest these fluorophores from natural systems could reap rich commercial benefit to bio-imaging industry. Silk cocoon biomaterial is one such example of natural system, which has acquired a fluorescent signature. The objective of this study is to develop simple, rapid, commercially viable technique to isolate silk cocoon membrane fluorophores and exploring the possibility of using them as fluorescent dye in bio-imaging. Here, we report an innovative water glass (Na2SiO3) based strategy to isolate the silk cocoon fluorophores. Isolated fluorophore is majorly quercetin derivatives and exhibited remarkable photo- and heat stability. Fluorescence and mass spectrometric analysis confirmed presence of a quercetin derivative. We further used this fluorophore to successfully label the silicate shell of diatom species Nitzschia palea.
The tumor microenvironment (TME) comprises a heterogeneous number and type of cellular and noncellular components that vary in the context of molecular, genomic and epigenomic levels. The genotypic diversity and plasticity within cancer cells are known to be affected by genomic instability and genome alterations. Besides genomic instability within the chromosomal linear DNA, an extra factor appears in the form of extrachromosomal circular DNAs (eccDNAs; 2–20 kbp) and microDNAs (200–400 bp). This extra heterogeneity within cancer cells in the form of an abundance of eccDNAs adds another dimension to the expression of procancer players, such as oncoproteins, acting as a driver for cancer cell survival and proliferation. This article reviews research into eccDNAs centering around cancer plasticity and hallmarks, and discusses these facts in light of therapeutics and biomarker development.
Valve interstitial cells are dispersed throughout the heart valve and play an important role in maintaining its integrity, function, and phenotype. While prior studies have detailed the role of external mechanical and biological factors in the function of the interstitial cell, the role of cell shape in regulating contractile function, in the context of normal and diseased phenotypes, is not well understood. Thus, the aim of this study was to elucidate the link between cell shape, phenotype, and acute functional contractile output. Valve interstitial cell monolayers with defined cellular shapes were engineered via constraining cells to micropatterned protein lines (10, 20, 40, 60 or 80µm wide). Samples were cultured in either normal or osteogenic medium. Cellular shape and architecture were quantified via fluorescent imaging techniques. Cellular contractility was quantified using a valve thin film assay and phenotype analyzed via western blotting, zymography, and qRT-PCR. In all pattern widths, cells were highly aligned, with maximum cell and nuclear elongation occurring for the 10μm pattern width. Cellular contractility was highest for the most elongated cells, but was also increased in cells on the widest pattern (80μm) that also had increased CX43 expression, suggesting a role for both elongated shape and increased cell-cell contact in regulating contractility. Cells cultured in osteogenic medium had greater expression of smooth muscle markers and correspondingly increased contractile stress responses. Cell phenotype did not significantly correlate with altered cell shape, suggesting that cellular shape plays a significant role in the regulation of valve contractile function independent of phenotype.
Background More than five million Americans suffer from heart valve disease annually, a condition that worsens cardiac function and gradually leads to heart failure if appropriate treatment is not performed on time. Currently no medication can cure heart valve disease, leaving surgical intervention as the only viable option for patients at late stages of cardiac valve disease. Tremendous efforts have been undertaken to elucidate how resident cells in the valves respond to pathological stimulation as well as the underlying mechanisms that regulate these responses, to identify potential therapeutic targets for non-surgical treatment of valvular heart disease. Results Cardiac valve interstitial cells (VICs) naturally reside in a complex three-dimensional environment under varying hemodynamics, which is difficult to replicate in vitro . As a result, most cell signaling studies in the field have traditionally been conducted on two-dimensional models or in the absence of hemodynamic forces. Previously, we reported the fabrication of a hydrogel scaffold that could be used to culture valve cells under dynamic mechanical stimulation in a valve-mimetic environment. This model, therefore appeared to be suitable for VIC signaling studies as it provided cells a three-dimensional environment with the ability to incorporate mechanical stretching stimulation. Utilizing this model, we investigated the possible role of fibroblast growth factor 1 and 2 (FGF1 and FGF2) via FGFR1 receptor signaling in regulating valve cell activation under physiological (10% stretch) and pathological (20% stretch) mechanical conditions as well as in mediating cell proliferation and metabolism via the Akt/mTOR pathways. We reported that 1) FGF1 and FGF2 treatment was able to maintain the quiescent phenotype of VICs; 2) Cells increased proliferation as determined by optical redox ratios under elevated cyclic stretch via Akt/mTOR pathways; and 3) FGF1 and 2 signaling via the FGFR1 reduced VIC proliferation and activation under elevated cyclic stretch conditions. Conclusions Overall, these results suggested that targeting FGFR1 receptor signaling may represent a possible therapeutic strategy for preventing heart valve disease progression. Electronic supplementary material The online version of this article (10.1186/s13036-019-0168-1) contains supplementary material, which is available to authorized users.
Calcific aortic valve disease (CAVD) is the most common form of valve disease where the only available treatment strategy is surgical valve replacement. Technologies for the early detection of CAVD would benefit the development of prevention, mitigation and alternate therapeutic strategies. Two-photon excited fluorescence (TPEF) microscopy is a label-free, non-destructive imaging technique that has been shown to correlate with multiple markers for cellular differentiation and phenotypic changes in cancer and wound healing. Here we show how specific TPEF markers, namely, the optical redox ratio and mitochondrial fractal dimension, correlate with structural, functional and phenotypic changes occurring in the aortic valve interstitial cells (VICs) during osteogenic differentiation. The optical redox ratio, and fractal dimension of mitochondria were assessed and correlated with gene expression and nuclear morphology of VICs. The optical redox ratio decreased for VICs during early osteogenic differentiation and correlated with biological markers for CAVD progression. Fractal dimension correlated with structural and osteogenic markers as well as measures of nuclear morphology. Our study suggests that TPEF imaging markers, specifically the optical redox ratio and mitochondrial fractal dimension, can be potentially used as a tool for assessing early CAVD progression in vitro. Calcific aortic stenosis or calcific aortic valve disease (CAVD) is a progressive disease involving multiple signaling pathways, endothelial dysfunction, cytokine infiltration, collagen remodeling, as well as lipid and calcium deposition 1-4. The symptoms and markers for CAVD manifest via both degenerative (apoptotic) and active (osteogenic) mechanisms 5,6. Aortic valve endothelial and interstitial cells differentiate into an osteoblast-like phenotype, the extracellular matrix becomes thicker and stiffer and calcium mineralization occurs throughout the tissue 7,8. Aortic stenosis and sclerosis have an increased prevalence in the elderly and contribute to a 50% elevated risk of infarction and other potentially fatal cardiovascular pathologies 2,9,10. Currently, valve replacement is the preferred treatment method, as other strategies, such as the retardation of calcific progression, prevention and early diagnosis, are non-existent 11. Diagnostic techniques like echocardiography, cardiac MRI and cardiac CT are widely used, but are only sensitive during later stages of the disease once there is tissue mineralization, and hemodynamic and geometric impairment 12,13. Aortic valve interstitial cells (VICs) are the primary cells in the heart valves and are involved in tissue maintenance, repair and remodeling 14. VICs exist in a quiescent state under healthy conditions and are activated due to injury or disease 15 , potentially differentiating into an osteogenic-like phenotype to potentiate calcification 2. Current in vitro biochemical techniques to assess CAVD are typically destructive as they involve cell lysis or fixation and do not facilitate the longitu...
Background Calcific aortic valve disease (CAVD) pathophysiology is a complex, multistage process, usually diagnosed at advanced stages after significant anatomical and hemodynamic changes in the valve. Early detection of disease progression is thus pivotal in the development of prevention and mitigation strategies. In this study, we developed a diet-based, non-genetically modified mouse model for early CAVD progression, and explored the utility of two-photon excited fluorescence (TPEF) microscopy for early detection of CAVD progression. TPEF imaging provides label-free, non-invasive, quantitative metrics with the potential to correlate with multiple stages of CAVD pathophysiology including calcium deposition, collagen remodeling and osteogenic differentiation. Methods Twenty-week old C57BL/6J mice were fed either a control or pro-calcific diet for 16 weeks and monitored via echocardiography, histology, immunohistochemistry, and quantitative polarized light imaging. Additionally, TPEF imaging was used to quantify tissue autofluorescence (A) at 755 nm, 810 nm and 860 nm excitation, to calculate TPEF 755–860 ratio (A860/525/(A755/460 + A860/525)) and TPEF Collagen-Calcium ratio (A810/525/(A810/460 + A810/525)) in the murine valves. In a separate experiment, animals were fed the above diets till 28 weeks to assess for later-stage calcification. Results Pro-calcific mice showed evidence of lipid deposition at 4 weeks and calcification at 16 weeks at the valve commissures. The valves of pro-calcific mice also showed positive expression for markers of osteogenic differentiation, myofibroblast activation, proliferation, inflammatory cytokines and collagen remodeling. Pro-calcific mice exhibited lower TPEF autofluorescence ratios, at locations coincident with calcification, that correlated with increased collagen disorganization and positive expression of osteogenic markers. Additionally, locations with lower TPEF autofluorescence ratios at 4 and 16 weeks exhibited increased calcification at later 28-week timepoints. Conclusions This study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology.
While the valvulopathic effects of serotonin (5HT) and angiotensin-II (Ang-II) individually are known, it was not clear how 5HT and Ang-II might interact, specifically in the context of the mechanobiological responses due to altered valve mechanics potentiated by these molecules. In this context, the hypothesis of this study was that increased serotonin levels would result in accelerated progression toward disease in the presence of angiotensin-II-induced hypertension. C57/BL6 J mice were divided into four groups and subcutaneously implanted with osmotic pumps containing: PBS (control), 5HT (2.5 ng/kg/min), Ang-II (400 ng/kg/min), and 5HT + Ang-II (combination). Blood pressure was monitored using the tail cuff method. Echocardiography was performed on the mice before surgery and every week thereafter to assess ejection fraction. After three weeks, the mice were sacrificed and their hearts excised, embedded and sectioned for analysis of the aortic valves via histology and immunohistochemistry. In separate experiments, porcine valve interstitial cells (VICs) were directly stimulated with 5HT (10 M), Ang-II (100 nM) or both and assayed for cellular contractility, cytoskeletal organization and collagen remodeling. After three weeks, average systolic blood pressure was significantly increased in the 5HT, Ang-II and combination groups compared to control. Echocardiographic analysis demonstrated significantly reduced ejection fraction in Ang-II and the combination groups. H&E staining demonstrated thicker leaflets in the combination groups, suggesting a more aggressive remodeling process. Picrosirius red staining and image analysis suggested that the Ang-II and combination groups had the largest proportion of thicker collagen fibers. VIC orientation, cellular contractility and collagen gene expression was highest for the 5HT + Ang-II combination treatment compared to all other groups. Overall, our results suggest that 5HT and Ang-II interact to result in significantly detrimental alteration of function and remodeling in the valve.
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