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Alhaji Osman Smith,1,2 Wen Ju,1– 3 Seyram Yao Adzraku,1,2 Lu wenyi,1,2 Chen Yuting,1,2 Jianlin Qiao,1– 3 Kailin Xu,1– 3 Lingyu Zeng1– 3 1Department of Blood Diseases Institute, Xuzhou Medical University, Xuzhou City, 221002, Jiangsu Province, People’s Republic of China; 2Department of Key Laboratory of the Bone Marrow Stem Cell, Xuzhou Medical University, Xuzhou City, 221002, Jiangsu Province, People’s Republic of China; 3Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou City, 221002, Jiangsu Province, People’s Republic of ChinaCorrespondence: Kailin Xu; Lingyu Zeng Tel +8615162166166; +8613685182368Email lihmd@163.com; zengly2000@163.comIntroduction: The extend to the clinical benefit of radiation therapy is the inability to eliminate only cancer cells and destroy normal cells such as microvascular endothelial in the vascular niche and turn induced-inflammasome signaling and cell death. These unfortunate injuries generated by ionizing radiation alter the therapeutic window and result in the re-occurrence of the malignancy. Therefore, we engaged in vitro studies by demonstrating radiation-induced inflammasome and cell death in endothelial cells.Methods: The microvascular endothelial cells were cultured in a sterile dish, then kept in a humidifier of 5% at 37°C for 12 hours/more to attain confluence, and exposed at a dose of 1.8Gy/min achieve the coveted amounts except for the control. The cells were harvested 24 hours post-irradiation.Results: Our findings indicate that gamma radiation activates the NOD-like receptor (NLR) family of NLRP1 and NLRP3 complex in microvascular endothelial cells. These complexes activate the inactive precursor of caspase-1, which cleaved to bioactive caspase − 1 and enhances the production of pro-inflammatory cytokines of interleukin-1β and interleukin-18 that induce the dependent pyroptotic, which results in the production of chemokines, tumor necrosis factor-alpha (TNF-α), and high-mobility group protein-1 (HMGB-1). We also discovered the radiation could directly prompt caspase − 1, which auto-cleaved to activate gasdermin D to potentiate pyroptosis independently.Discussion: Overall, these findings suggested that reducing the unfavorable effect of radiation injuries could be challenging since gamma radiation induces the microvascular endothelial cells to cell death and activates the inflammasome signaling via different pathways.Keywords: gamma radiation, microvascular endothelial cells, ECs, NOD-like receptor, NLR, inflammasome, pyroptosis

Smith AO, Ju W, Adzraku SY, et al. J Inflamm Res. 2021;14:3277–3288 Following a review of the paper post-publication, the authors realized they had mistakenly duplicated the immunofluorescence on Figure 6 during the figure preparation. On page 3286, the corrected Figure 6 is shown in Download Article: Figure 6 Gamma radiation directly activates Caspase-1 dependent pathway in microvascular endothelial cells. Abbreviation: NS, not significant. Notes: (A) Immunofluorescence images of cleaved caspase −1 in microvascular endothelial cells. (B) The mean fluorescence intensity of cleaved caspase −1 in microvascular endothelial cells in the graph was relatively quantified by image J, which shows a dose-dependent increase in the expression of cleaved caspase-1. The results were representative of 6 independent experiments of four repeats (N=4) (mean ± SD) *P

Background: Family violence homicide (FVH) is a major public health and social problem in Australia. FVH trend rates are key outcomes that determine the effectiveness of current management practices and policy directions. Data source–related methodological problems affect FVH research and policy and the reliable measurement of homicide trends. Objective: This study aimed to determine data reliability and temporal trends of Victorian FVH rates and sex and relationship patterns. Method: FVH rates per 100,000 persons in Victoria were compared between the National Coronial Information System (NCIS), Coroners Court of Victoria (CCoV) Homicide Register, and the National Homicide Monitoring Program (NHMP). Trends for 2001–2017 were analysed using Joinpoint regression. Crude rates were determined by sex and relationship categories using annual frequencies and Australian Bureau of Statistics population estimates. Results: NCIS closed FVH cases totalled 360, and an apparent downward trend in the FVH rate was identified. However, CCoV and NHMP rates trended upwards. While NCIS and CCoV were case-based, NHMP was incident-based, contributing to rate variations. The NCIS-derived trend was particularly impacted by unavailable case data, potential coding errors and entry backlog. Neither CCoV nor NHMP provided victim-age in their public domain data to enable age-adjusted rate comparison. Conclusion: Current datasets have limitations for FVH trend determination; most notably lag times for NCIS data. Implications: This study identified an indicative upward trend in FVH rates in Victoria, suggesting insufficiency of current management and policy settings for its prevention and control. [ABSTRACT FROM AUTHOR]

Polycystic kidney disease (PKD) is an important cause of kidney failure, but treatment options are limited. While later stages of the disease have been extensively studied, mechanisms driving the initial conversion of kidney tubules into cysts are not understood. To identify genes with the potential to promote cyst initiation, we deleted polycystin-2 (Pkd2) in mice and surveyed transcriptional changes before and immediately after cysts developed. We identified 74 genes which we term cyst initiation candidates (CICs). To identify conserved changes with relevance to human disease we compared these murine CICs to single cell transcriptomic data derived from patients with PKD and from healthy controls. Tumor-associated calcium signal transducer 2 (Tacstd2) stood out as an epithelial-expressed gene with elevated levels early in cystic transformation that further increased with disease progression. Human tissue biopsies and organoids show that TACSTD2 protein is low in normal kidney cells but is elevated in cyst lining cells, making it an excellent candidate for mechanistic exploration of its role in cyst initiation. While TACSTD2 has not been studied in PKD, it has been studied in cancer where it is highly expressed in solid tumors while showing minimal expression in normal tissue. This property is being exploited by antibody drug conjugates that target TACSTD2 for the delivery of cytotoxic drugs. Our finding that Tacstd2/TACSTD2 is prevalent in cysts, but not normal tissue, suggests that it should be explored as a candidate for drug development in PKD. More immediately, our work suggests that PKD patients undergoing TACSTD2-directed treatment for breast and urothelial cancer should be monitored for kidney effects., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A provisional patent has been filed: US Application No.: 63/589,316 THERAPY FOR POLYCYSTIC KIDNEY DISEASE, (Copyright: © 2024 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bakey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Early detection of firearm discharge has become increasingly critical for situational awareness in both civilian and military domains. The ability to determine the location and model of a discharged firearm is vital, as this can inform effective response plans. To this end, several gunshot audio datasets have been released that aim to facilitate gunshot detection and classification of a discharged firearm based on acoustic signatures. However, these datasets often suffer from a lack of variety in the orientations of recording devices around the source of the gunshot. Additionally, these datasets often suffer from the absence of proper time synchronization, which prevents the usage of these datasets for determining the Direction of Arrival (DoA) of the sound. In this paper, we present a multi-firearm, multi-orientation time-synchronized audio dataset collected in a semi-controlled real-world setting - providing us a degree of supervision - using several edge devices positioned in and around an outdoor firing range., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Inc.)

Tumor immunotherapy, one of the efficient therapies in cancers, has been called to the scientific community's increasing attention lately. Among them, immune checkpoint inhibitors, providing entirely new modalities to treat cancer by leveraging the patient's immune system. They are first-line treatments for varieties of advanced malignancy, such as melanoma, gastrointestinal tumor, esophageal cancer. Although immune checkpoint inhibitors (ICIs) treatment has been successful in different cancers, drug resistance and relapses are common, such as in colorectal cancer. Therefore, it is necessary to improve the efficacy of immune checkpoint therapy for cancer patients who do not respond or lowly response to current treatments. N6-methyladenosine (m6A), as a critical regulator of transcript expression, is the most frequently internal modification of mRNA in the human body. Recently, it has been proposed that m6A epigenetic modification is a potential driver of tumor drug resistance. In this report, we will briefly outline the relevant mechanisms, general treatment status of immune checkpoint inhibitors in colorectal cancer, how m6A epigenetic modifications regulate the response of ICIs in CRC and provide new strategies for overcoming the resistance of ICIs in CRC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tong, Wei, Smith and Huang.)

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people and is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The mechanisms by which polycystin mutations induce cyst formation are not well understood, however pro-proliferative signaling must be involved for tubule epithelial cell number to increase over time. We recently found that the stress-activated mitogen-activated protein kinase (MAPK) pathway c-Jun N-terminal kinase (JNK) pathway is activated in cystic disease and genetically removing JNK reduces cyst growth driven by a loss of Pkd2. This review covers the current state of knowledge of signaling in ADPKD with an emphasis on the JNK pathway., Competing Interests: Disclosures The authors have no conflicts to disclose.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The most proximal effects of Pkd mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The c-Jun N-terminal kinase (JNK) pathway promotes proliferation and is activated in acute and chronic kidney diseases. Using a mouse model of cystic kidney disease caused by Pkd2 loss, we observe JNK activation in cystic kidneys and observe increased nuclear phospho c-Jun in cystic epithelium. Genetic removal of Jnk1 and Jnk2 suppresses the nuclear accumulation of phospho c-Jun, reduces proliferation and reduces the severity of cystic disease. While Jnk1 and Jnk2 are thought to have largely overlapping functions, we find that Jnk1 loss is nearly as effective as the double loss of Jnk1 and Jnk2. Jnk pathway inhibitors are in development for neurodegeneration, cancer, and fibrotic diseases. Our work suggests that the JNK pathway should be explored as a therapeutic target for ADPKD., Competing Interests: The authors have declared that no competing interests exist.

In the bone marrow microenvironment, endothelial cells (ECs) play a pivotal role in regulating the production of both growth and inhibiting factors. They are held together by adherence molecules that interact with hematopoietic progenitor cells. The study of ECs in the hematopoietic stem cell niche is limited due to the lack of efficient protocols for isolation. In this protocol, we developed a two-step approach to extract bone marrow endothelial cells (BMECs) to unlock the challenges researchers face in understanding the function of the endothelial vascular niche in in-vitro studies., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Background: In the bone marrow microenvironment (BM), endothelial cells are individual cells that form part of the sinusoidal blood vessels called the "bone marrow endothelial-vascular niche." They account for less than 2% of the bone marrow cells. They play essential functions by generating growth and inhibitory factors that promote the hematopoietic stem cells (HSCs) regulation. In response to inflammatory stimuli, the BMECs increase in proliferation to maintain the blood vessels' integrity within the BM. The inflammatory response releases cytokines such as tumor necrosis factor-alpha (TNF-α) that promote vascular endothelial cells' expansion and upregulation of adhesion molecules (ICAM-1 and VCAM-1, respectively) in the BM. However, the evaluation of mouse BMECs in the bone marrow microenvironment is scared by a lack of mouse bone marrow endothelial cell primary culture METHODS: Two steps approach for isolation of bone marrow endothelial cells (BMECs) from mice. In brief, the bone marrow cells extracted from the mice long bones were cultured overnight with Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and antibiotics to separate between marrow-derived adherent and non-adherent cells. The floating cells were discarded, and the adhered section detached with accutase and BMECs selected using CD31 microbeads. The isolated BMECs were cultured in a dish pre-coated with rat-tail collagen type 1 with endothelial cells medium supplement with growth factors. The cells were verified by confocal microscopy for morphology and tube formation by matrigel assay. We validate the cells' purity by flow cytometry, RT-qPCR, immunofluorescence staining, and immunoblotting by established BMEC markers, PECAM-1, VE-cadherin, vascular endothelial cell growth factor receptor-2 (VEGFR2), CD45, E-selectin, and endothelial selectin adhesion molecule (ESAM). Lastly, we characterize BMEC activation with recombinant TNF-α., Results: Our method clearly defined the cells isolated have the characteristics of BMECs with the expression of CD31, VE-cadherin, E-selectin, VEGFR-2, and ESAM. The cells' response to TNF-α indicates its inflammatory function by increasing proliferation and upregulation of adhesion molecules., Conclusions: This study outline a simple new technique of isolating mouse BMEC primary culture and a suitable method to evaluate the function and dysregulation of BMEC in in vitro studies using mouse models.

Background: Low back pain is one of the leading causes of disability worldwide. Exercise therapy is widely recommended to treat persistent non-specific low back pain. While evidence suggests exercise is, on average, moderately effective, there remains uncertainty about which individuals might benefit the most from exercise., Methods: In parallel with a Cochrane review update, we requested individual participant data (IPD) from high-quality randomised clinical trials of adults with our two primary outcomes of interest, pain and functional limitations, and calculated global recovery. We compiled a master data set including baseline participant characteristics, exercise and comparison characteristics, and outcomes at short-term, moderate-term and long-term follow-up. We conducted descriptive analyses and one-stage IPD meta-analysis using multilevel mixed-effects regression of the overall treatment effect and prespecified potential treatment effect modifiers., Results: We received IPD for 27 trials (3514 participants). For studies included in this analysis, compared with no treatment/usual care, exercise therapy on average reduced pain (mean effect/100 (95% CI) -10.7 (-14.1 to -7.4)), a result compatible with a clinically important 20% smallest worthwhile effect. Exercise therapy reduced functional limitations with a clinically important 23% improvement (mean effect/100 (95% CI) -10.2 (-13.2 to -7.3)) at short-term follow-up. Not having heavy physical demands at work and medication use for low back pain were potential treatment effect modifiers-these were associated with superior exercise outcomes relative to non-exercise comparisons. Lower body mass index was also associated with better outcomes in exercise compared with no treatment/usual care. This study was limited by inconsistent availability and measurement of participant characteristics., Conclusions: This study provides potentially useful information to help treat patients and design future studies of exercise interventions that are better matched to specific subgroups. PROTOCOL PUBLICATION: https://doi.org/10.1186/2046-4053-1-64., Competing Interests: Competing interests: MvT and the members of the Chronic LBP IPD Meta-analysis Group are investigators of the individual trials included in the IPD data set., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Epithelial cells lining the ducts and tubules of the kidney nephron and collecting duct have a single non-motile cilium projecting from their surface into the lumen of the tubule. These organelles were long considered vestigial remnants left as a result of evolution from a ciliated ancestor, but we now recognize them as critical sensory antennae. In the kidney, the polycystins and fibrocystin, products of the major human polycystic kidney disease genes, localize to this organelle. The polycystins and fibrocystin, through an unknown mechanism, monitor the diameter of the kidney tubules and regulate the proliferation and differentiation of the cells lining the tubule. When the polycystins, fibrocystin or cilia themselves are defective, the cell perceives this as a pro-proliferative signal, which leads to tubule dilation and cystic disease. In addition to critical roles in preventing cyst formation in the kidney, cilia are also important in cystic and fibrotic diseases of the liver and pancreas, and ciliary defects lead to a variety of developmental abnormalities that cause structural birth defects in most organs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: Healthcare organizations need to rapidly adapt to new technology, policy changes, evolving payment strategies, and other environmental changes. We report on the development and application of a structured methodology to support technology and process improvement in healthcare organizations, Systematic Iterative Organizational Diagnostics (SIOD). SIOD was designed to evaluate clinical work practices, diagnose technology and workflow issues, and recommend potential solutions., Materials and Methods: SIOD consists of five stages: (1) Background Scan, (2) Engagement Building, (3) Data Acquisition, (4) Data Analysis, and (5) Reporting and Debriefing. Our team applied the SIOD approach in two ambulatory clinics and an integrated ambulatory care center and used SIOD components during an evaluation of a large-scale health information technology transition., Results: During the initial SIOD application in two ambulatory clinics, five major analysis themes were identified, grounded in the data: putting patients first, reducing the chaos, matching space to function, technology making work harder, and staffing is more than numbers. Additional themes were identified based on SIOD application to a multidisciplinary clinical center. The team also developed contextually grounded recommendations to address issues identified through applying SIOD., Discussion: The SIOD methodology fills a problem identification gap in existing process improvement systems through an emphasis on issue discovery, holistic clinic functionality, and inclusion of diverse perspectives. SIOD can diagnose issues where approaches as Lean, Six Sigma, and other organizational interventions can be applied., Conclusion: The complex structure of work and technology in healthcare requires specialized diagnostic strategies to identify and resolve issues, and SIOD fills this need., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Medical Informatics Association.)

Murine bone marrow-derived macrophages (M0) and M1- and M2-polarized macrophages are being widely used as a laboratory model for polarized macrophages related molecular mechanism analysis. Gene expression analysis based on reference gene normalization using RT-qPCR was a powerful way to explore the molecular mechanism. But little is known about reference genes in these cell models. So, the goal of this study was to identify reference genes in these types of macrophages. Candidate reference genes in murine bone marrow-derived and polarized macrophages were selected from microarray data using Limma linear model method and evaluated by determining the stability value using five algorithms: BestKeeper, NormFinder, GeNorm, Delta CT method, and RefFinder. Finally, the selected stable reference genes were validated by testing three important immune and inflammatory genes (NLRP1, IL-1β, and TNF-α) in the cell lines. Our study has clearly shown that Ubc followed by Eef1a1 and B2m respectively were recognized as the three ideal reference genes for gene expression analysis in murine bone marrow-derived and polarized macrophages. When three reference genes with strong different stability were used for validation, a large variation of a gene expression level of IL-1β, TNF-α and NLRP1 were obtained which provides clear evidence of the need for careful selection of reference genes for RT-qPCR analysis. Normalization of mRNA expression level with Ubc rather than Actb or Gusb by qPCR in macrophages and polarized macrophages is required to ensure the accuracy of the qPCR analysis.

Platelets contract forcefully after their activation, contributing to the strength and stability of platelet aggregates and fibrin clots during blood coagulation. Viscoelastic approaches can be used to assess platelet-induced clot strengthening, but they require thrombin and fibrin generation and are unable to measure platelet forces directly. Here, we report a rapid, microfluidic approach for measuring the contractile force of platelet aggregates for the detection of platelet dysfunction. We find that platelet forces are significantly reduced when blood samples are treated with inhibitors of myosin, GPIb-IX-V, integrin α IIb β 3, P2Y 12 , or thromboxane generation. Clinically, we find that platelet forces are measurably lower in cardiology patients taking aspirin. We also find that measuring platelet forces can identify Emergency Department trauma patients who subsequently require blood transfusions. Together, these findings indicate that microfluidic quantification of platelet forces may be a rapid and useful approach for monitoring both antiplatelet therapy and traumatic bleeding risk.

Endothelial cells (ECs) could express some important cytokines and signal molecules which play a key role in normal hematopoiesis and repopulation. Busulfan-induced vascular endothelial injury is an important feature after hematopoietic stem cell transplantation (HSCT). But the molecular mechanism of how the injured ECs affect hematopoietic reconstruction is still unknown. It is possibly through modulation of the change of some gene expression. RT-qPCR is one of the most popular methods used to accurately determine gene expression levels, based on stable reference gene (RG) selection from housekeeping genes. So our aim is to select stable RGs for more accurate measures of mRNA levels during Busulfan-induced vascular endothelial injury. In this study, 14 RGs were selected to investigate their expression stability in ECs during 72 hours of EC injury treated with Busulfan. Our results revealed extreme variation in RG stability compared by five statistical algorithms. ywhaz and alas1 were recognized as the two idlest RGs on account of the final ranking, while the two most usually used RGs (gapdh and actb) were not the most stable RGs. Next, these data were verified by testing signalling pathway genes ctnnb1, robo4, and notch1 based on the above four genes ywha, alas1, gapdh, and actb. It shows that the normalization of mRNA expression data using unstable RGs greatly affects gene fold change, which means the reliability of the biological conclusions is questionable. Based on the best RGs used, we also found that robo4 is significantly overexpressed in Busulfan-impaired ECs. In conclusion, our data reaffirms the importance of RGs selection for the valid analysis of gene expression in Busulfan-impaired ECs. And it also provides very useful guidance and basis for more accurate differential expression gene screening and future expanding biomolecule study of different drugs such as cyclophosphamide and fludarabine-injured ECs.

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms., (Published by Elsevier Inc.)

We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life.

This study aimed to form microvessels in fibrin gels, which is of interest both for studying the fundamental cell-matrix interactions as well as for tissue engineering purposes, and to align the microvessels, which would provide natural inlet and outlet sides for perfusion. The data reported here demonstrate the formation of highly interconnected microvessels in fibrin gel under defined medium conditions and the ability to align them using two methods, both of which involved anchoring the gel at both ends to constrain the cell-induced compaction. The first method used only defined medium and resulted in moderate alignment. The second method used defined and serum-containing media sequentially to achieve high levels of microvessel alignment., (© 2013.)

An important advance using in vitro EC tube morphogenesis and maturation models has been the development of systems using serum-free defined media. Using this approach, the growth factors and cytokines which are actually necessary for these events can be determined. The first model developed by our laboratory was such a system where we showed that phorbol ester was needed in order to promote survival and tube morphogenesis in 3D collagen matrices. Recently, we have developed a new system in which the hematopoietic stem cell cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and stromal derived factor-1α (SDF-1α) were added in conjunction with FGF-2 to promote human EC tube morphogenesis in 3D collagen matrices under serum-free defined conditions. This new model using SCF, IL-3, SDF-1α, and FGF-2 also works well following the addition of pericytes where EC tube formation occurs, pericytes are recruited to the tubes, and vascular basement membrane matrix assembly occurs following EC-pericyte interactions. In this chapter, we describe several in vitro assay models that we routinely utilize to investigate the molecular requirements that are critical to EC tube formation and maturation events in 3D extracellular matrix environments.

Cardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralog, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development, as well as postnatal vessel growth., (Copyright © 2012 Elsevier Inc. All rights reserved.)