Name: demonstrating a linear relationship between vascular endothelial growth factor and luteinizing hormone in kidney cortex extracts
Uploaded: 2020-01-22T14:00:00
Description: Watch this Scientific Journal Video about Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts at JoVE.com

The authors thank Scholl&#39;s Slaughterhouse (Blissfield, MI) for providing the bovine and porcine kidneys. No grant funding was utilized for this study.

No live or experimental animals were used for this study.
1. Tissue Handling
<ol>
	<li>Procure bovine and porcine whole kidneys immediately after slaughter from an abattoir. Transport on ice to the laboratory.</li>
	<li>Upon arrival at laboratory, rinse kidneys with 50 mL of ice-cold phosphate buffered saline (PBS). Repeat this step 2x to remove blood completely.</li>
	<li>Keep kidneys on ice (or refrigerated) until further extraction.</li>
</ol>
2. Dissection of Kidney...

<ol>
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Z., Sigler, R., Muthusamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitreous+Levels+of+Luteinizing+Hormone+and+VEGF+are+Strongly+Correlated+in+Healthy+Mammalian+Eyes.">Vitreous Levels of Luteinizing Hormone and VEGF are Strongly Correlated in Healthy Mammalian Eyes.</a> Current Eye Research. 43 (8), 1041-1044 (2018).</li><li>Babitha, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luteinizing+hormone,+insulin+like+growth+factor-1,+and+epidermal+growth+factor+stimulate+vascular+endothelial+growth+factor+production+in+cultured+bubaline+granulosa+cells.">Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells.</a> General and Comparative Endocrinology. 198, 1-12 (2014).</li><li>Trau, H. 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Z., Sigler, R., Muthusamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+Signaling+by+the+Luteinizing+Hormone+Receptor+Reduces+Ocular+VEGF+and+Retinal+Vascularization+during+Mouse+Eye+Development.">Elimination of Signaling by the Luteinizing Hormone Receptor Reduces Ocular VEGF and Retinal Vascularization during Mouse Eye Development.</a> Current Eye Research. 43 (10), 1286-1289 (2018).</li><li>Hipkin, R. W., Sanchez-Yague, J., Ascoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+luteinizing+hormone/chorionic+gonadotropin+(LH/CG)+receptor+precursor+in+a+human+kidney+cell+line+stably+transfected+with+the+rat+luteal+LH/CG+receptor+complementary+DNA.">Identification and characterization of a luteinizing hormone/chorionic gonadotropin (LH/CG) receptor precursor in a human kidney cell line stably transfected with the rat luteal LH/CG receptor complementary DNA.</a> Molecular Endocrinology. 6 (12), 2210-2218 (1992).</li><li>Lei, Z. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+luteinizing+hormone/human+chorionic+gonadotropin+receptor+gene.">Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene.</a> Molecular Endocrinology. 15 (1), 184-200 (2001).</li><li>Schrijvers, B. F., Flyvbjerg, A., De Vriese, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vascular+endothelial+growth+factor+(VEGF)+in+renal+pathophysiology.">The role of vascular endothelial growth factor (VEGF) in renal pathophysiology.</a> Kidney International. 65 (6), 2003-2017 (2004).</li><li>Apaja, P. M., Aatsinki, J. T., Rajaniemim, H. J., Petaja-Repo, U. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+mature+luteinizing+hormone+receptor+in+rodent+urogenital+and+adrenal+tissues+is+developmentally+regulated+at+a+posttranslational+level.">Expression of the mature luteinizing hormone receptor in rodent urogenital and adrenal tissues is developmentally regulated at a posttranslational level.</a> Endocrinology. 146 (8), 3224-3232 (2005).</li><li>Ondruschka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-mortem+in+situ+stability+of+serum+markers+of+cerebral+damage+and+acute+phase+response.">Post-mortem in situ stability of serum markers of cerebral damage and acute phase response.</a> International Journal of Legal Medicine. 133 (3), 871-881 (2019).</li><li>Swain, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+post-mortem+interval:+A+comparison+between+cerebrospinal+fluid+and+vitreous+humour+chemistry.">Estimation of post-mortem interval: A comparison between cerebrospinal fluid and vitreous humour chemistry.</a> Journal of Forensic and Legal Medicine. 36, 144-148 (2015).</li><li>Thompson, C. S., Traynor, I. M., Fodey, T. L., Faulkner, D. V., Crooks, S. R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+method+for+the+detection+of+residues+of+amphenicol+antibiotics+in+bovine,+ovine+and+porcine+kidney+by+optical+biosensor.">Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor.</a> Talanta. 172, 120-125 (2017).</li><li>Konstantinou, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-Linked+Immunosorbent+Assay+(ELISA).">Enzyme-Linked Immunosorbent Assay (ELISA).</a> Methods in Molecular Biology. 1592, 79-94 (2017).</li><li>Levesque, B. 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Procuring kidneys from the abattoir immediately after animal death is the key to success in this methodology. This is the main advantage of utilizing organs from cows and pigs instead of human cadavers. There is usually at least a 12-24 h delay from the time of death until human cadaver organs are procured. Because the chemical composition of bodily organs significantly changes within 2 h post-mortem15,16, VEGF-studies in human cadaver kidneys may not reflect rea...

Vascular endothelial growth factor A (VEGF-A), helps to regulate angiogenesis and vascular permeability in the kidney and other organs1,2 (hereafter, VEGF-A will be referred to as VEGF). VEGF levels in the kidney are under tight homeostatic control. When renal VEGF levels are elevated or depressed, the kidney can malfunction. For example, within 3 weeks after birth, mice with podocyte-specific heterozygosity for VEGF develop endotheliosis and bloodless glomeruli (i.e, renal lesions seen in human preeclampsia), and end-stage kidney failure occurs in these heterozygotes by 3 months of age. Podocyte-specific homozygotic knockouts die from hydrops and kidney failure within 1 day of birth3,4.
On the other hand, overexpression of renal VEGF causes proteinuria and glomerular hypertrophy3,4. For example, transgenic rabbits that overexpress VEGF exhibit progressive proteinuria with increased glomerular filtration rates in early stages of nephropathy, followed by decreased glomerular filtration rates in later stages3. Diabetic nephropathy, a major cause of end-stage renal disease in diabetic adults, is strongly associated with VEGF dysregulation2,5. A great deal of attention has been paid to the role of hypoxia in inducing VEGF expression under pathologic conditions5. However, the factors governing VEGF under physiologic conditions (both in the kidney and other organs) are not well-understood2,6. Identifying these factors (except for oxygen) that are involved in physiologic and pathologic VEGF regulation is an important undertaking.
Luteinizing hormone (LH), a pro-angiogenic hormone, helps regulate physiologic VEGF expression in reproductive organs such as the ovary and testis7,8. Previous studies have provided evidence that LH also helps regulate VEGF in non-reproductive organs, such as the eyes6,9,10. LH receptors are found in the medulla and cortex of the kidney11,12. Of note, kidney tubular epithelial cells, as well as the LH receptor, express VEGF11,12,13,14. Taking these two observations together, we hypothesized that LH also helps regulate VEGF expression in the kidney13,14. To provide evidence of this LH/VEGF relationship, the presented protocol aims to show that LH levels are able to predict VEGF levels in the kidney. Many previous VEGF-related investigations involving the kidney have used lower order mammal models (i.e., rodents and rabbits)2. To translate this work to the human body, the study examines the relationship between VEGF and LH in higher order mammals (here, bovine and porcine models). To carry out this objective, total protein lysate was prepared from the cortex region of bovine and porcine kidneys.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine LH ELISA Kit</td>
			<td>MyBiosource, San Diego, CA.</td>
			<td>MBS700951</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine VEGF-A ELISA Kit</td>
			<td>MyBiosource, San Diego, CA.</td>
			<td>MBS2887434</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific Inc, Columbus, OH.</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine LH ELISA Kit</td>
			<td>MyBiosource, San Diego, CA.</td>
			<td>MBS009739</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine VEGF-A ELISA</td>
			<td>Ray Biotech, Norcross, GA.</td>
			<td>ELP-VEGFA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher Scientific Inc, Columbus, OH.</td>
			<td>89901</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating a linear relationship between vascular endothelial growth factor and luteinizing hormone in kidney cortex extracts

Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic nephropathy, are associated with VEGF dysregulation in the kidney. The factors that govern VEGF under physiologic conditions in the kidney are not well-understood. Luteinizing hormone (LH), a pro-angiogenic hormone, helps regulate physiologic VEGF expression in reproductive organs. Given that LH receptors are found in the kidney, we, at Zietchick Research Institute, hypothesized here that LH also helps regulate VEGF expression in the kidney as well. To provide evidence, we aimed to show that LH levels are able to predict VEGF levels in the mammalian kidney. Most VEGF-related investigations involving the kidney have used lower order mammals as models (i.e., rodents and rabbits). To translate this work to the human body, it was decided to examine the relationship between VEGF and LH in higher order mammals (i.e., bovine and porcine models). This protocol uses the total protein lysate from the kidney cortex. Keys to this method&#39;s success include procurement of kidneys from slaughterhouse animals immediately after death as well as normalization of analyte levels (in the kidney extract) by total protein. This study successfully demonstrates a significant linear relationship between LH and VEGF in both bovine and porcine kidneys. The results are reproducible in two different species. The study provides supporting evidence that the use of kidney extracts from cows and pigs are an excellent, economical, and abundant resource for the study of renal physiology, particularly for examining the correlation between VEGF and other analytes.

The mean and median levels of LH and VEGF by animal type and by sex are shown in Table 1. After verifying normality of data by Kolmogorov-Smirnov Testing of normality, linear regression models were utilized to examine the relationship between LH and VEGF. LH was found to be a strong and significant predictor of VEGF in both bovine and porcine kidneys (bovine kidney model: n = 7, R2 = 0.86, p = 0.002; porcine kidney model: n = 7; R2 = 0.66, p = 0.025)...

Watch this Scientific Journal Video about Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts at JoVE.com

Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts

Presented here is a protocol for utilizing a cortical kidney extract preparation and total protein normalization to demonstrate the correlation between vascular endothelial growth factor and luteinizing hormone in the mammalian kidney.

Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic ...

Our protocol observes that the use of kidney extracts from cows and pigs are an excellent and economical resource for the study of renal physiology, particularly examining the correlation between vascular endothelial growth factor and other analytes. It has always been a challenge to correlate VEGF, an essential growth factor in organ angiogenesis, with other proteins. This technique allows us to examine the association between VEGF and analytes such as hormones.This will certainly advance our base of knowledge regarding this important growth factor. This method may be useful for correlating other analytes in kidney cortical extracts besides the ones mentioned in this video. Visual demonstration will help others to obtain reproducible results without any sampling errors.The use of fresh tissue is essential to the success of this technique. It is important to obtain fresh whole kidneys immediately after slaughter from an animal. Always transfer the tissue to the laboratory on ice.Use sterile scissors, forceps, a knife, and Petri dishes to dissect kidneys and excise the required tissue portion. Gently cut the kidney in half along the sagittal plane and cut a piece of tissue from the cortex region in the center of the kidney. Mince the tissue into small pieces with the knife and transfer it into a microfuge tube with one milliliter of ice cold 1X RIPA lysis extraction buffer.Label the tube with specific sample details and place it on ice. Use a handheld homogenizer with a sterile probe to homogenize the tissue for one to two minutes until no chunks are visible, then immediately centrifuge the tube at 9, 600 times g for five minutes at four degrees Celsius. After centrifugation, prepare separate aliquots of the sample for ELISA and total protein analysis to avoid multiple freeze/thaw cycles.Prior to starting the assay, bring all reagents and the assay plate to room temperature removing the excess wells and storing them at four degrees Celsius until further use. Dilute the 20X wash buffer to 300 milliliters with double distilled water and set up the blank wells without any solution. Add 50 microliters of standard or sample and another 50 microliters of horseradish peroxidase conjugate to each well, then immediately add 50 microliters of antibody solution to each well and seal the plate.Mix the plate and incubate it at 37 degrees Celsius for one hour. After the incubation, wash each well with 200 microliters of wash buffer. Add 50 microliters of substrate A and substrate B to each well, mix the plate gently by tapping on the side, then seal and incubate it in the dark for 15 minutes at 37 degrees Celsius.Add 50 microliters of stop solution to each well. Gently tap the plate and read the absorbance at 450 nanometers with a spectrophotometer. Prepare the reagents and wash buffer as previously described, then add 100 microliters of standard or sample to each well, seal the plate, mix well, and incubate it for two hours at 37 degrees Celsius.Remove the liquid in each well and add 100 microliters of detection reagent A.Seal the plate and incubate it for another hour. Next, wash each well with 400 microliters of wash buffer, then add 90 microliters of substrate solution to each well, gently tap the plate, and incubate it for another hour at 37 degrees Celsius. After the last incubation, add 50 microliters of stop solution to each well, gently tap the plate, and read the absorbance at 450 nanometers with a spectrophotometer.Estimate the total protein of the bovine and porcine kidney extracts with a standard BSA assay and use this estimate to normalize the LH and VEGF-A-ELISA results. The mean and median levels of LH and VEGF were calculated for both animals and sexes. After verifying normality of data, linear regression models were utilized to examine the relationship between LH and VEGF.LH was found to be a strong and significant predictor of VEGF in both bovine and porcine kidneys. When attempting this procedure, it is important to remember to sample identical areas in each tissue. A similar procedure can be used to extract any type of tissue.Remember to normalize analytes by total protein extract of whatever tissue you use.

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The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
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The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...