Name: Video: Author Spotlight: Advances in Quantifying Microvascular Density in Aging Murine Lungs
Uploaded: 2025-01-03T12:40:00.000+00:00
Duration: 10 min
Description: 2.4K Views. Sichuan University. Here, we describe a simple and economical protocol to perform unbiased quantification of pulmonary microvascular density for whole mice lung tissue using single staining of Isolectin B4.

The authors express their gratitude for the invaluable support received from the public experimental platform at West China School of Pharmacy. Special appreciation is extended to Wendong Wang for providing critical and highly valuable advice on pathology. This research has been made possible through the funding from the Science and Technology Department of Sichuan Province (grants 2023NSFSC0130 and 2023NSFSC1992) and &#34;the Fundamental Research Funds for the Central Universities&#34; to TJ.

All the experiments were carried out following the ethical guidelines of the Sichuan University Animal Research Committee (No K2023023).
1. Preparation of paraffin sections for mouse lung lobes
<ol>
	<li>Acquire lung tissue from the mouse.
	<ol>
		<li>Choose male C57Bl/6 mice at 6 weeks and 16 months to assess the microvascular density in mouse lungs across different age groups.</li>
		<li>Administer 100 mg/kg of pentobarbital sodium via intraperitoneal injection in mice. Euthanize the mice by cervical dislocation after anesthesia. Open the chest cavity and obtain lung tissue.</li>
	</ol>
	</li>
	<li>Prepare paraffin sections.
	<ol>
		<li>Fix the tissue by placing it in a 4% paraformaldehyde solution that is 10 times the volume of the tissue to preserve its morphology and structure and prevent degradation. 
		NOTE: A recent new method was developed to inflate air during vascular perfusion-fixation in murine lungs. This method is reported to preserve better morphology and location of the airway, alveolar cells, and interstitium, making them more suitable for lung histologic studies. This method is recommended for laboratories with the corresponding equipment and consumables with in-house-made instruments8.</li>
		<li>Cut the lung lobe to determine the embedding direction (Figure 1).
		<ol>
			<li>After 3 h of fixation, separate the five pulmonary lobes (under direct observation of the eyes) and embed the cranial, middle, and accessory lobes of the right lung separately, with sections oriented in the direction of the vertical lobe-to-bronchus connection.</li>
			<li>Cut the caudal lobe of the right lung into 2 pieces at 4 mm from left to right in the direction perpendicular to the lung lobe-bronchus connection, and embed the slices in a wax block with the cut surface facing upward.</li>
			<li>Make three cuts in the left lung lobe. Make the first cut transversely above the main bronchial junction, 2 mm below the top. Make the second cut transversely above the main bronchial junction, 5 mm below the top. Make the third cut at the end of the lobe, 8 mm below the top. Discard the uppermost piece of tissue and encase the remaining three pieces in a wax block with the cut surface facing upward. 
			NOTE: When embedding multiple lung pieces together, a microscope wiping paper can be used to wrap them before placing them in a tissue embedding box to continue the dehydration process.</li>
			<li>Re-fix the tissue in the fixative for 24 h.</li>
		</ol>
		</li>
		<li>Place the tissue block in a container and rinse it under flowing water for 15 min.</li>
		<li>Sequentially place the tissue in the following solutions: 65% ethanol for 30 min, 75% ethanol for 30 min, 85% ethanol for 30 min, 95% ethanol I for 60 min, 95% ethanol II for 60 min, absolute ethanol I for 60 min, absolute ethanol II for 60 min, 70% ethanol for 120 min, 80% ethanol for 120 min, 90% ethanol for 120 min, 95% ethanol for 120 min, absolute ethanol I for 120 min, and absolute ethanol II for 120 min.</li>
		<li>Place the tissue in xylene I for 30 min and xylene II for 30 min.</li>
		<li>Place the tissue in soft wax below 54 &#176;C for 1 h, hard wax I at 58 &#176;C for 1 h, and hard wax II at 58 &#176;C for 30 min.</li>
		<li>Select a suitable mold size, fill it with an appropriate amount of liquid paraffin, and use forceps to position the tissue in the embedding frame in the center of the mold in the correct direction. Place the mold and tissue in the freezing area of the embedding machine and gently press the tissue with forceps.</li>
		<li>When the paraffin starts to whiten slightly, quickly position the uncovered embedding frame on top of the mold and perform the second wax injection, sealing the holes at the bottom of the embedding frame. Transfer the entire mold to the freezing table for molding.</li>
		<li>After securing the wax block onto the slicer and revealing the smooth and flat cut surface through rough and fine trimming, rotate the microtome&#39;s wheel crank to cut slices with a thickness of 4 &#181;m.</li>
		<li>Smoothly place the removed sections onto the water surface in sequential order. Once the sections are fully extended, separate the broken pieces at the junctions between each section.</li>
		<li>Tilt a glass slide underneath the section and, upon contact, slowly and evenly lift the slide out of the water surface in a vertical motion. The sections will adhere to the glass slide.</li>
	</ol>
	</li>
</ol>
2. Immunofluorescence staining for detection of pulmonary microvasculature
<ol>
	<li>Place the paraffin-embedded slices in a 65 &#176;C oven for 60 min.</li>
	<li>Place the slices on a slide rack and carry out the following steps in staining jars: immerse xylene 1, xylene 2, and xylene 3 for 15 min each, followed by immersion in xylene/ethanol (1:1) for 5 min, 100% ethanol #1, 100% ethanol #2, 85% ethanol, 75% ethanol, and distilled water for 5 min each.</li>
	<li>Dilute the modified sodium citrate antigen retrieval solution 50-fold with deionized water. Preheat the microwave on high power until boiling, then place the slide in the antigen extract. Allow the slide to boil in the antigen extract for 15 min and then cool naturally to room temperature.</li>
	<li>Wash the slides twice with PBS for 5 min each. Permeabilize with 0.25% Triton twice for 10 min each.</li>
	<li>Encircle the tissue with an immunohistochemistry pen and incubate that with 5% BSA at room temperature (RT) for 1 h. Wash the slides once with PBS for 5 min.</li>
	<li>Perform Alexa-647 Fluor conjugated IB4 and DAPI staining.
	<ol>
		<li>Keep the IB4 on ice and dilute it 1:50 with 1% BSA. Remove excess water around the circle, add 50-100 &#181;L of the diluted IB4 to each tissue section, and incubate overnight at 4 &#176;C (from this step onwards, perform all procedures in the dark).</li>
		<li>Wash the slides three times with PBS for 5 min each. Permeabilize with 0.25% Triton for 10 min.</li>
	</ol>
	</li>
	<li>Dilute DAPI (10 &#181;g/mL) with PBS at a ratio of 1:10 and add 50-100 &#181;L to each tissue section at RT for 5 min.</li>
	<li>Wash the slides three times with PBS for 5 min each.</li>
	<li>Apply a drop of anti-fade mounting medium to the slides, avoiding light exposure. Air dry the slide, then observe and capture images of the nucleus and target signals under a fluorescence microscope.</li>
</ol>
3. Quantification of pulmonary microvascular density
<ol>
	<li>Acquire images.
	<ol>
		<li>Observe lung microvascular density signals and DAPI signals under a 10x objective for each lung lobe in both the young and old age groups.</li>
		<li>Standardize the light source intensity and exposure time for each channel based on the observation results. Capture the dual-channel images covering the entire area of each lung lobe. Save the image in ND2 format with two channels (Figure 2).</li>
	</ol>
	</li>
	<li>Quantify using Image J (Supplementary Figure 1).
	<ol>
		<li>Launch ImageJ software.</li>
		<li>Import indicated images to ImageJ, then proceed to load and select them. Split the channels accordingly. Load a file in ND2 format for both DAPI and IB4 channels.</li>
		<li>Navigate to Analyze &#62; Set Scale &#62; Configure the Image Parameters as 0.48&#160;&#181;m/pixel (depending on the photography parameters) &#62; Global &#62; OK.</li>
		<li>Set the same display range to adjust the image presentation effect. Select Image&#62; Adjust &#62; Brightness/Contrast &#62; Set. Set display range for each of the two channels separately, IB4: 0-10000; DAPI: 0-20000.</li>
		<li>To eliminate the influence of background signals, proceed with the following steps: Click Process &#62; Math &#62; Subtract; Subtract the background signal based on the signal values detected by the Magic Wand tool on the background, IB4: 3000; DAPI:300.</li>
		<li>To select the threshold value that best aligns with the original image, go to Image &#62; Duplicate.</li>
		<li>Select both original and duplicate Image &#62; Type &#62; 8 bit.</li>
		<li>Select an appropriate threshold value to precisely identify the lung microvasculature. Click on Image &#62; Adjust &#62; Threshold &#62; Intermodes&#160;(choose the most realistic threshold against the original diagram), tick &#62; Dark Background &#62; Apply.</li>
		<li>Eliminate IB4 positive signals from large blood vessels in the mouse lung, including arteries and veins: Use the Magic Wand tool to select larger blood vessels compared to the duplicated image, then click Delete&#160;(repeat that until the IB4 positive signals in the endothelial cells of larger blood vessels are removed). 
		NOTE: IB4 staining signals are detected in endothelial cells of arteries and veins in the mouse lung. To achieve a specific examination of pulmonary microvasculature occurrences, it is recommended to uniformly remove the signal foci in endothelial cells of larger blood vessels.</li>
		<li>Count the number of target signals, click Analyze &#62; Set Measurements, and tick Area, Limit to Threshold, and Display Label. Then select Analyze Particles and set Size: 0-infinity; Circularity: 0.00-1.00; Show: Overly Masks; tick Summarize and click OK.</li>
		<li>Choose Summarize Results &#62; File &#62; Save as.</li>
	</ol>
	</li>
	<li>Data analysis and statistics
	<ol>
		<li>Match and organize the number of IB4 positive foci and DAPI positive foci, respectively, at the same image into a unified table.</li>
		<li>Summarize the total number of IB4 positive foci and DAPI positive foci for each lobe in indicated young or age groups.
		<ol>
			<li>Calculate the percentage of IB4 positive foci (%) by dividing the total number of IB4 positive foci by the total number of DAPI staining foci in each lobe by different groups. Present the number of IB4 positive foci, the number of DAPI staining foci, and the percentage of IB4 positive foci (%) as shown in Figure 3A.</li>
		</ol>
		</li>
		<li>Launch the data analysis software and create a new column table. 
		NOTE: GraphPad Prism 9 was used here for statistical analysis.</li>
		<li>Sequentially name them from Group A to Group J as &#34;Young cranial lobe&#34;, &#34;Old cranial lobe&#34;, &#34;Young middle lobe&#34;, &#34;Old middle lobe&#34;, &#34;Young caudal lobe&#34;, &#34;Old caudal lobe&#34;, &#34;Young accessory lobe&#34;, &#34;Old accessory lobe&#34;, &#34;Young left lobe&#34;, &#34;Old left lobe&#34;. Then, input the corresponding IB4 positive foci (%) into the table (Supplementary Table 1).</li>
		<li>Utilize unpaired t-tests for pairwise comparison to assess significant differences in the corresponding regions between the two age groups. Conduct a total of five comparisons for five lobes.</li>
		<li>Create a graph for result visualization (Figure 3B). Opt for a bar chart and include statistical analysis in the figure.</li>
	</ol>
	</li>
</ol>

Assessing Pulmonary Microvasculature with Isolectin B4 Staining in Mice

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The authors declare that they have no competing interests.

The study of pulmonary microvascular density holds significant implications for understanding pulmonary physiological processes and also for defining biomarker(s) for respiratory diseases. The pulmonary circulation boasts an extensive capillary surface area enveloped by a slender layer of endothelial cells. The harmonious juxtaposition of these cells and alveolar epithelial cells gives rise to a fragile alveolar-capillary membrane specifically designed to facilitate the intricate process of gas exchange...

Endothelial cells (ECs) are a special type of cell located on the inner lining of blood vessels, covering the entire arterial and venous tree and playing a crucial role in maintaining the stability of blood vessels and organs1. The lungs are highly vascularized organs and play essential physiological and pathological roles in the lungs, such as forming the vascular wall, regulating blood flow, facilitating gas exchange, modulating inflammatory responses, controlling platelet activity, secreting regulatory substances involved in vascular growth, repair, and maintaining coagulation balance.
Lung microvascular endothelial cells (LMECs) are specific endothelial cells of pulmonary tissue, particularly in the microvasculature (capillaries) of the lungs, distinguishing them from the more generalized arterial and venous endothelial cells in the lungs. These cells have various functions, including regulating vascular tone, controlling vascular permeability, participating in the regulation of inflammatory responses, and regulating thrombus formation. They play a crucial role in pulmonary circulation, regulating gas exchange and the transport of nutrients, and are involved in various physiological and pathological processes related to the lungs, likely for aging2. Moreover, the abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction3. Employing the conventional endothelial cell marker CD31 and spatial localization (specifically, the peripheral regions of the lungs), Larissa L.&#160;et al. observed a significant decrease in microvascular endothelial cell density in aged mice (18 months old) compared to their younger counterparts (4 months old)4. In the context of the pulmonary pathology associated with asthma, Makoto H. et al. demonstrated a substantial increase in vessel induction in bronchial biopsy specimens stained with anti-collagen IV from asthmatic patients in comparison to control subjects5. Recently, by introducing the techniques of transmission and scanning electron microscopy, Maximilian A. et al. reported a notable increase in the numeric density of features related to intussusceptive and sprouting angiogenesis in patients who died from Covid-19 or Influenza A (H1N1)6. Evidently, the abnormal microvascular genesis is linked with pulmonary dysfunction. However, there is currently no simple, economical method available for quantifying changes in microvascular density.
In murine models, the lungs are conventionally segmented into five distinct lobes: right cranial, right middle, right caudal, left cranial, and left caudal. Each lobe exhibits unique characteristics in terms of size, shape, location, and likely vascularization, contributing to efficient gas exchange and potentially synergistic regulation of pulmonary circulation. However, to the best of our knowledge, no methodologies account for the differences among these lung lobes when investigating Lung microvascular changes.
This study presents a new method for sectioning lobules in mice, utilizing IB4, a well-defined marker of lung micro-endothelial cells7, for unbiased quantitative assessment of pulmonary microvascular density. This innovative approach addresses the need for a more comprehensive understanding of microvascular alterations in murine lungs by considering the distinct properties of individual lobes of mice. As a demonstration, in aging&#160;mice, a significant reduction in pulmonary 
microvascular density is observed specifically&#160;within both the caudal lobe and left lobe.&#160;The protocol underscores the importance of integrating lobe-specific analyses into investigations of changes in the microvascular landscape of murine lungs. Notably, this method provides valuable research references for investigators seeking a comprehensive understanding of both physiological and pathological progression of lung developments and lesions, extending beyond angiogenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td>Tissue Fixative</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole</td>
			<td>MCE</td>
			<td>HY-D0814</td>
			<td>Nucleic Dyes</td>
		</tr>
		<tr>
			<td>Alexa-647 Fluor Conjugated Isolectin B4</td>
			<td>Thermo</td>
			<td>I32450</td>
			<td>Binding Microvessels</td>
		</tr>
		<tr>
			<td>Anti-fluorescent Tablet Sealer</td>
			<td>Abcam</td>
			<td>AB104135</td>
			<td>Sample Fixation</td>
		</tr>
		<tr>
			<td>Antigen Repair Fluid</td>
			<td>Biosharp</td>
			<td>BL151A</td>
			<td>Repair of Antigenic Sites</td>
		</tr>
		<tr>
			<td>Biopsy Cassette</td>
			<td>ActivFlo</td>
			<td>39LC-500-1</td>
			<td>Fixing and Positioning Tissue Samples</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>B2064-50G</td>
			<td>Sealing Solution</td>
		</tr>
		<tr>
			<td>Cold Plate</td>
			<td>Leica</td>
			<td>HistoCore Arcadia H&#160;</td>
			<td>Freezing Samples</td>
		</tr>
		<tr>
			<td>Constant Temperature Electric Drying Oven</td>
			<td>Taisite</td>
			<td>101-0AB</td>
			<td>High Temperature Repair</td>
		</tr>
		<tr>
			<td>Disposable Microtome Blade</td>
			<td>Leica</td>
			<td>14035838383</td>
			<td>Cutting Tissue Samples to Prepare Sections</td>
		</tr>
		<tr>
			<td>Embedding Molds</td>
			<td>Shitai</td>
			<td>26155166627</td>
			<td>Fixing Tissue Samples</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Kelong</td>
			<td>CAS 64-17-5</td>
			<td>Tissue Dehydration Solution</td>
		</tr>
		<tr>
			<td>Heated Paraffin Embedding Station</td>
			<td>Leica</td>
			<td>EG1150</td>
			<td>Embedding Tssue Samples in Paraffin</td>
		</tr>
		<tr>
			<td>HistoCore Water Bath</td>
			<td>Leica</td>
			<td>HI1220</td>
			<td>Flatten and Fix Tissue Samples</td>
		</tr>
		<tr>
			<td>ImageJ (Fiji)&#160;</td>
			<td>NIH</td>
			<td>1.54f</td>
			<td>Quantitative Tool</td>
		</tr>
		<tr>
			<td>Immunohistochemistry Pens</td>
			<td>Biosharp</td>
			<td>BC004</td>
			<td>Water-blocking Agent</td>
		</tr>
		<tr>
			<td>Medical Forceps</td>
			<td>Shanghai Medical Equipment</td>
			<td>N/A</td>
			<td>Grasping, Manipulating, or Moving tissue samples</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Ts2</td>
			<td>Imaging Device</td>
		</tr>
		<tr>
			<td>Mounting Media</td>
			<td>Jiangyuan</td>
			<td>Tasteless</td>
			<td>Fixing and Preserving Tissue Sections</td>
		</tr>
		<tr>
			<td>Paraffin Wax</td>
			<td>SCHLEDEN</td>
			<td>80200-0014</td>
			<td>Fixing Tissue Structure</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Beyotime</td>
			<td>C0221A</td>
			<td>Wash Buffer</td>
		</tr>
		<tr>
			<td>Pentobarbital Sodium</td>
			<td>Beijing Chemical Reagent Company</td>
			<td>Q/H82-F158-2002</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Rotary Microtome</td>
			<td>Biobase</td>
			<td>Bk-2258</td>
			<td>Preparing Slices</td>
		</tr>
		<tr>
			<td>Sterile Scissors</td>
			<td>Shanghai Medical Equipment</td>
			<td>N/A</td>
			<td>segmenting Tissue Samples</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Shanghai Medical Equipment</td>
			<td>N/A</td>
			<td>Cutting Tissue Samples</td>
		</tr>
		<tr>
			<td>Triton&#160;</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td>Permeabilization Solution</td>
		</tr>
		<tr>
			<td>Wash-Free Slide</td>
			<td>PLATINUM PRO</td>
			<td>PRO-04</td>
			<td>Fixing Samples for Staining</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SUM</td>
			<td>XK13-011-00031</td>
			<td>Tissue De-waxing Solution</td>
		</tr>
	</tbody>
</table>

quantifying pulmonary microvascular density in mice across lobules

The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood flow regulation, and gas exchange. In murine models, lung lobes exhibit significant differences in size, shape, location, and vascularization, yet existing methods lack consideration for these variations when quantifying microvascular density. This limitation hinders the comprehensive study of lung microvascular dysfunction and the potential remodeling of microvasculature circulation across different lobules. Our protocol addresses this gap by employing two sectioning methods to quantify pulmonary microvascular density changes, leveraging the size, shape, and distribution of airway branches across distinct lobes in mice. We then utilize Isolectin B4 (IB4) staining to label lung microvascular endothelial cells on different slices, followed by unbiased microvascular density analysis using the freely available software ImageJ. The results presented here highlight varying degrees of microvascular density changes across lung lobules with aging, comparing young and old mice. This protocol offers a straightforward and cost-effective approach for unbiased quantification of pulmonary microvascular density, facilitating research on both physiological and pathological aspects of lung microvasculature.

To distinguish between the lesions in the main bronchi and small airway branches, it is crucial to ensure that the continuous structure of lesions in these two types of airways is observed. This can be achieved by following the cutting and embedding procedures outlined in Figure 1. Given the numerous lung lobes in mice, which are oriented in various directions and possess a mesh-like structure, they are more susceptible to collapse compared to other solid tissues. To achieve a clear and intu...

Author Spotlight: Advances in Quantifying Microvascular Density in Aging Murine Lungs

Here, we describe a simple and economical protocol to perform unbiased quantification of pulmonary microvascular density for whole mice lung tissue using single staining of Isolectin B4.

Quantifying Pulmonary Microvascular Density in Mice Across Lobules

The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood ...

quantifying-pulmonary-microvascular-density-in-mice-across-lobules

Research

JoVE Journal

Biology

2.4K Views.  Sichuan University. Here, we describe a simple and economical protocol to perform unbiased quantification of pulmonary microvascular density for whole mice lung tissue using single staining of Isolectin B4.

Video: Author Spotlight: Advances in Quantifying Microvascular Density in Aging Murine Lungs

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.

We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...