This work was supported by a Nuclear Research and Development Program of the National Research Foundation of Korea (NRF) grant funded by the Korean government (No. 2017M2A2A6A02019904).

All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals under protocols approved by the Korea Institute of Radiological and Medical Sciences Animal Studies Committee. All reagents and solvents were purchased and used without further purification. NOTA-RGD-peptides were prepared according to literature methods15.
CAUTION: 68Ga emits both positron and gamma rays. All experiments, including direct or indirect contact with radioactive substances, must be undertaken by trained and permitted personnel only. When handling radioactive materials, proper protective equipment, shielding, radiation dosimeter badge and rings, and a survey meter should be used.
1. Radiolabeling RGD-peptides with 68GaCl3
Note: 68Ga (t1/2 = 68 min, &#946;+ = 89%, and EC = 11%) was obtained from the 68Ga/68Ge generator.
<ol>
	<li>Elute the 68GaCl3 from the generator with 4 mL of 0.05 M HCl.</li>
	<li>Purge with nitrogen gas at 80 &#176;C for 30 min to dry 68GaCl3 (333 kBq, 1 mL) in a 5 mL reaction vial.</li>
	<li>Add a solution of RGD-peptide (100 &#181;g)in 1 M sodium acetate (100 &#181;L, pH 5 - 6) to the reaction vial containing 68GaCl3 from step 1.2.</li>
	<li>Heat the reaction mixture at 80 &#176;C for 5 min. Then, cool it down to room temperature.</li>
	<li>Purify the crude product with high-performance liquid chromatography (HPLC). Use the following system: a C-18 column, a flow rate of 0.5 mL/min, a gradient slope of acetonitrile of 1.17%/min (5% - 40% in 30 min), and elution components: A = 0.1% trifluoroacetic acid (TFA) in acetonitrile, B = 0.1% TFA in water. 
	NOTE: The HPLC is equipped with a photodiode array detector and a radioactivity detector. The 68Ga-RGD-peptide was collected at a retention time of 12.5 min (Figure 2).</li>
	<li>Purify the resulting 68Ga-RGD-peptide using a solid phase extraction system.
	<ol>
		<li>Pass the solution through a C18 reverse-phase cartridge and wash with 2 mL of saline.</li>
		<li>Elute 68Ga-RGD-peptide with 0.7 mL of 95% ethanol. Remove the solvent at 80 &#176;C under nitrogen gas for 20 min and reconstitute with phosphate-buffered saline (PBS) before use.</li>
		<li>Filter the radiolabeled product through a 0.22 &#181;m sterile filter and formulate in 1 mL of sterile saline solution.</li>
	</ol>
	</li>
	<li>Check the radiochemical yield by radio-thin-layer chromatography (TLC).
	<ol>
		<li>Spot 1 &#181;L on an instant thin layer chromatography plate (ITLC, 10 cm in length). Develop the plate in a chamber containing the eluent (aqueous 0.1 M citric acid, pH 5.0) until 9 cm away from the spot. 
		NOTE: The retention factor for 68Ga-RGD-peptide is 0 and the retention factor for unreacted 68Ga3+ is 1.</li>
	</ol>
	</li>
	<li>Calculate the final specific activity from the ratio of radioactivity corresponding to the non-radioactivity as MBq/nmol. 
	NOTE: After the injection of 100 &#181;L of the formulated 68Ga-RGD-peptide to HPLC, the amount of non-radioactive component was calculated from the standard calibration curve using nonradioactive Ga-RGD-peptide.</li>
</ol>
2. In Vitro Cellular Uptake
Note: Uppsala 87 Malignant Glioma (U87MG) human glioblastoma cells were grown in Dulbecco&#39;s modified Eagle&#39;s media (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were grown in 150 mm dishes at 37 &#176;C in a humidified atmosphere of 5% CO2. Cells were harvested or split by trypsinization: 0.25% (w/v) trypsin and 0.02% (w/v) ethylenediaminetetraacetic acid (EDTA) in PBS at 37 &#176;C for 3 - 5 min.
<ol>
	<li>Seed U87MG cells into 6-well plates at a density of 1 x 106 cells/well.</li>
	<li>Incubate the cells with 68Ga-RGD-peptide (111 kBq) at 37 &#176;C for 30, 60, 90, and 120 min. Prepare samples in triplicate.</li>
	<li>Wash the cells 2x with 2 mL of PBS and harvest by trypsinization. Use 0.25% (w/v) trypsin and 0.02% (w/v) ethylenediaminetetraacetic acid (EDTA) in PBS at 37 &#176;C for 3 - 5 min.</li>
	<li>Collect the cell suspension (500 &#181;L) and measure in a &#947;-counter.</li>
	<li>Calculate the percent uptake of the compound by the cells by % (counts in cells/total counts).</li>
</ol>
3. In Vitro Serum Stability
<ol>
	<li>Add 500 &#181;L of freshly prepared mouse serum, 500 &#181;L of human serum, and 500 &#181;L of PBS. Incubate the mixture at 37 &#176;C for 2 h.</li>
	<li>Evaluate by ITLC at the specified time intervals (30, 60, 90, and 120 min). Spot 1 - 2 &#181;L aliquot of the mixture to the ITLC plate (mobile phase: 0.1 M citric acid). Develop the plate as in step 1.7. 
	NOTE: 68Ga3+ is expected to move with the solvent front, whereas the labeled compound will remain at the origin.</li>
</ol>
4. Determination of Lipophilicity
<ol>
	<li>Add 68Ga-RGD-peptide (3.7 MBq, 3.7 &#181;L) to the octanol-PBS system (1:1, v/v, total 1 mL).</li>
	<li>Mix the vials vigorously for 5 min at room temperature and centrifuge at 10,000 x g for 5 min at room temperature.</li>
	<li>Take 100 &#181;L samples from each layer and measure the radioactivity with a &#947;-counter. The reported log P value is based on the average of three samples.</li>
</ol>
5. Tumor Model
Note: BALB/c nude mice (6 - 8 weeks old, female, n = 23) were used for this study. The mice were subsequently used for PET studies (n = 3) and biodistribution (n = 20) when the tumor volumes reached 200 - 300 mm3 (1 - 2 weeks after implantation).
<ol>
	<li>Load tumor cells into 28 G, 1/2 inch insulin syringes.</li>
	<li>Inject U87MG cells (5 x 106) in 100 &#181;L of PBS into the left arm region.</li>
	<li>Anesthetize the mouse with 2% isoflurane in oxygen gas during cell injection.
	<ol>
		<li>Ensure that the mouse has been anesthetized by the loss of the pedal withdrawal reflex following pinching with forceps between the toes of the right hind foot. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
</ol>
6. In Vivo Quantification of &#945;v&#946;3 Integrin Using PET
<ol>
	<li>Anesthetize the mice with 2% isoflurane in oxygen.
	<ol>
		<li>Ensure that the mouse has been anesthetized by the loss of the pedal withdrawal reflex following pinching with forceps between the toes of the right hind foot. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Place the head in the center of the PET gantry.</li>
	<li>Intravenously administer the 68Ga-RGD-peptide solution (7.4 MBq, 200 &#181;L) to the xenograft mouse model via the tail vein for 1 min.</li>
	<li>At the same time, perform a PET scan in list mode (dynamic scan) for 150 min. 
	NOTE: The raw PET data were reconstructed by a user-defined time frame (i.e., every 30 min). After the PET scan, a micro-computed tomography (CT) scan (50 kVp of X-ray, 0.16 mA) was conducted for attenuation correction.</li>
</ol>
7. Ex Vivo Biodistribution
<ol>
	<li>Inject 68Ga-RGD-peptide (0.37 MBq, 200 &#181;L) into the tail vein of the xenograft mouse model. Anesthetize the mouse with 2% isoflurane in oxygen gas during the injections. 
	Note: BALB/c nude mice, as described in section 5, were divided into four groups and sacrificed at different time points (n = 5 per group).</li>
	<li>Wake the mice immediately after the administration of 68Ga-RGD-peptide and sacrifice them at 30, 60, 90, and 120 min postinjection with carbon dioxide euthanasia. 
	NOTE: The tissues of interest were extracted. Selected targets were the blood, muscle, heart, lung, liver, spleen, stomach, intestine, kidneys, bone, and tumor.</li>
	<li>Weigh the tissue and measure the radioactivity with a &#947;-counter. 
	NOTE: Results were expressed as the percentage injected dose per gram of tissue (% ID/g).</li>
</ol>

<ol>
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J., 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=Preparation+and+functional+evaluation+of+RGD-modified+proteins+as+&#945;v&#946;3+integrin+directed+therapeutics.">Preparation and functional evaluation of RGD-modified proteins as &#945;v&#946;3 integrin directed therapeutics.</a> Bioconjugate Chemistry. 13 (1), 128-135 (2002).</li><li>Garanger, E., 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=New+multifunctional+molecular+conjugate+vector+for+targeting,+imaging,+and+therapy+of+tumors.">New multifunctional molecular conjugate vector for targeting, imaging, and therapy of tumors.</a> Molecular Therapy. 12 (6), 1168-1175 (2005).</li><li>Dijkgraaf, I., 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=PET+imaging+of+&#945;v&#946;3+integrin+expression+in+tumours+with+68Ga-labelled+mono-,+di-+and+tetrameric+RGD+peptides.">PET imaging of &#945;v&#946;3 integrin expression in tumours with 68Ga-labelled mono-, di- and tetrameric RGD peptides.</a> European Journal of Nuclear Medicine and Molecular Imaging. 38 (1), 128-137 (2011).</li><li>Liu, Z., 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=68Ga-labeled+cyclic+RGD+dimers+with+Gly3and+PEG4linkers:+Promising+agents+for+tumor+integrin+&#945;v&#946;3+PET+imaging.">68Ga-labeled cyclic RGD dimers with Gly3and PEG4linkers: Promising agents for tumor integrin &#945;v&#946;3 PET imaging.</a> European Journal of Nuclear Medicine and Molecular Imaging. 36 (6), 947-957 (2009).</li><li>Li, Z. 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The authors have nothing to disclose.

In the present study, we introduced a protocol to prepare a radiopeptide targeting &#945;v&#946;3 integrin and its biological evaluation. Traditional drug development involves a complicated procedure. It requires a large quantity of reference material and a relatively long evaluation time. Although the suggested methodology cannot replace the delicate evaluation process, this system can be used for screening purposes. This proposed system would considerably reduce the time and cost.
<p class="jo...

Angiogenesis is a biological process that is characterized by the development of new blood vessels. Among many angiogenetic factors, &#945;v&#946;3 integrin is associated with invasiveness, because the integrin is highly expressed in angiogenic tumor vessels but is absent in normal tissue1.
Radiolabeled receptor-binding peptides with the arginine glycine aspartate (RGD) domain, which has a high affinity toward &#945;v&#946;3 integrin receptors, are considered promising angiogenesis imaging agents2,3,4,5,6,7. Several radiopharmaceuticals have been created for PET and its biological properties have been validated in various animal models8,9,10,11. In terms of a radionuclide, 68Ga has several advantages over other radioisotopes. Firstly, it has a high accessibility for users and is economically advantageous because a cyclotron is not required. Secondly, 68Ga-based radiopharmaceuticals produce high spatial resolution compared with single-photon emission computed tomography (SPECT), allowing more accurate quantification. Lastly, the 67.71 minutes half-life of 68Ga may be sufficient for the preparation of small peptides or proteins.
To produce a stable complex with 68Ga, many chelators have been developed. Representative chelators are 1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), diethylenetriaminepentaacetic acid (DTPA), and N,N&#39;-di(2-hydroxybenzyl)ethylenediamine-N,N&#39;-diacetic acid (HBED). NOTA has been reported to form a highly stable complex with 68Ga (log stability constant 30.98)12,13,14.
The purpose of the present study is to provide a concise protocol for the development of a new radiopeptide (Figure 1). As an example, we prepare 68Ga-labeled RGD-peptides and present methods for the biological evaluation of these analogues in a xenograft model.

<table border="0" cellpadding="0" cellspacing="0" style="width:775px;" width="775">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:231px;">68Ga/68Ge generator</td>
			<td style="width:187px;">ITG Company</td>
			<td style="width:119px;">-</td>
			<td style="width:239px;">10 mCi&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hydrogen chloride solution</td>
			<td style="width:187px;">Sigma-aldrich</td>
			<td>84429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium acetate</td>
			<td style="width:187px;">Sigma-aldrich</td>
			<td style="width:119px;">S2889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">C18 reverse-phase cartridge</td>
			<td style="width:187px;">Waters</td>
			<td style="width:119px;">WAT020515</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:231px;">0.22-&#956;m sterile filter</td>
			<td style="width:187px;">Milllipore</td>
			<td style="width:119px;">SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Radio-TLC scanner</td>
			<td style="width:187px;">Bioscan</td>
			<td style="width:119px;">AR2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">ITLC paper</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:119px;">SGI001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Citric acid</td>
			<td style="width:187px;">Sigma-aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:231px;">HPLC</td>
			<td style="width:187px;">Waters</td>
			<td style="width:119px;">-</td>
			<td style="width:239px;">Waters 1525 system containing binary pump, photo diode array (Waters 2998), radioactivity detector (Raytest, Gabi)</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:231px;">C-18 colunm</td>
			<td style="width:187px;">Develosil</td>
			<td style="width:119px;">HG31546150W</td>
			<td>ODS-HG-5, 150 &#8569; 4.6 mm, 5&#956;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Acetonitrile</td>
			<td style="width:187px;">J.T. Baker</td>
			<td style="width:119px;">14-650-359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trifluoroacetic acid</td>
			<td style="width:187px;">Sigma-aldrich</td>
			<td style="width:119px;">302031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dulbecco&#39;s modified Eagle media&#160;</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td style="width:119px;">11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td style="width:119px;">16000044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">T175 flasks&#160;</td>
			<td style="width:187px;">Corning</td>
			<td>CLS431080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">penicillin-streptomycin</td>
			<td style="width:187px;">Thermo fisher scientific</td>
			<td style="width:119px;">15240112</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">&#947;-counter</td>
			<td style="width:187px;">Perkin Elmer</td>
			<td style="width:119px;">-</td>
			<td style="width:239px;">1480 Wizard 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Insunlin syringe</td>
			<td style="width:187px;">Becton Dickinson</td>
			<td style="width:119px;">326105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Synringe pump</td>
			<td style="width:187px;">Harvard Apparatus</td>
			<td style="width:119px;">70-4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">micro-PET/CT</td>
			<td style="width:187px;">Siemens Inveon</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparing a 68ga-labeled arginine glycine aspartate (rgd)-peptide for angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in angiogenic tumor endothelial cells, where it typically has a low concentration. This specific expression of &#945;v&#946;3 makes it a valid biomarker for antiangiogenic and imaging drugs. As a functional imaging modality, positron emission tomography (PET) provides information about biochemical and physiological changes in vivo, due to its unique high sensitivity at the nanomolar scale. Hence, radiometal-based PET radiopharmaceuticals have received great attention for the non-invasive quantification of tumor angiogenesis. This paper provides a systemic protocol to prepare a new radiometal-labeled peptide for the evaluation of angiogenesis. This protocol contains information about radiochemical reliability, lipophilicity, cell uptake, serum stability, and pharmacokinetic properties. The 68Ga-RGD-peptide is one of the representative PET ligands toward &#945;v&#946;3 integrin. Here, we introduce a protocol to prepare a 68Ga-RGD-peptide and the evaluation of its biological efficacy.

The chelation of 68GaCl3 with the NOTA-RGD-peptide was straightforward, and the radiolabeling yield was 99%. Reaction impurities were successfully removed as shown in Figure 2. The radiochemical purity of 68Ga-RGD-peptide was greater than 99%, and specific activity at the end of the synthesis was 90 - 130 MBq/nmol (Figure 3).
The cell up...

Watch this Scientific Journal Video about Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis at JoVE.com

Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

Preparing a 68Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

The &#945;v&#946;3 integrin is a heterodimeric adhesion molecule involved in tumor cell migration and angiogenesis. The integrin is overexpressed in ...

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Research

JoVE Journal

Medicine

6.9K Views.  Korea Institute of Radiological and Medical Sciences. The αvβ3 integrin is a type of adhesion protein that is highly expressed on activated endothelial cells undergoing angiogenesis. Thus, evaluating the integrity of the integrin is of great interest in oncology. Here, we introduce a method to prepare 68Ga-labeled radiopeptides and a method to assess its biological effectiveness.

Video: Preparing a 68Ga-labeled Arginine Glycine Aspartate RGD-peptide for Angiogenesis

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among the most important players in the development and evolution of stroke-evoked brain edema is the hormone arginine-vasopressin and its receptors, V1a and V2. Recently, the V1a and V2 receptor blocker conivaptan has been attracting attention as a potential drug to reduce brain edema after stroke. However, animal models which involve conivaptan applications in stroke research need to be modified based on feasible routes of administration. Here the outcomes of 48 hr continuous intravenous (IV) are compared with intraperitoneal (IP) conivaptan treatments after experimental stroke in mice. We developed a protocol in which middle cerebral artery occlusion was combined with catheter installation into the jugular vein for IV treatment of conivaptan (0.2 mg) or vehicle. Different cohorts of animals were treated with 0.2 mg bolus of conivaptan or vehicle IP daily. Experimental stroke-evoked brain edema was evaluated in mice after continuous IV and IP treatments. Comparison of the results revealed that the continuous IV administration of conivaptan alleviates post-ischemic brain edema in mice, unlike the IP administration of conivaptan. We conclude that our model can be used for future studies of conivaptan applications in the context of stroke and brain edema.

Our studies have revealed that the beneficial effects of conivaptan are dependent on the method of delivery after experimental stroke in mice. We have developed a research protocol for delivery of the receptor blocker via IV catheter on stroke-evoked brain edema formation in mice.

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Preparing a ⁶⁸Ga-labeled Arginine Glycine Aspartate (RGD)-peptide for Angiogenesis

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A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

Angiogenesis in the Ischemic Rat Lung

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease