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• W2081167583 abstract "Therapeutic revascularization with either exogenous angiogenic growth factors or vascular cells has yet to demonstrate efficacy in the clinic. Injection of angiogenic growth factors often produces unstable and abnormal blood vessels. Blood vascular networks derived from implanted endothelial cells persist only transiently due to the insufficient recruitment of perivascular cells. We hypothesize that a combination of the two approaches may act synergistically to yield a better result. To enhance the recruitment of perivascular cells, human umbilical vein endothelial cells were genetically modified to overexpress platelet-derived growth factor (PDGF)-BB. PDGF-BB overexpression promoted both proliferation and migration of perivascular precursor cells (10T1/2 cells) in vitro. When mock-infected endothelial cells were implanted alone in vivo, they formed transient blood vascular networks that regressed by day 30. PDGF-BB overexpression enhanced the survival of endothelial cells in vivo. However, the PDGF-BB-expressing vessel network failed to establish patent blood flow. Co-implantation of PDGF-BB-overexpressing endothelial cells with 10T1/2 cells paradoxically resulted in the rapid regression of the vascular networks in vivo. PDGF-BB stimulated the expression of both chemokine (C-C motif) ligand 2 (CCL2) and CCL7 in 10T1/2 cells and led to the increased accumulation of macrophages in vivo. These results suggest a potential negative interaction between angiogenic growth factors and vascular cells; their use in combination should be carefully tested in vivo for such opposing effects. Therapeutic revascularization with either exogenous angiogenic growth factors or vascular cells has yet to demonstrate efficacy in the clinic. Injection of angiogenic growth factors often produces unstable and abnormal blood vessels. Blood vascular networks derived from implanted endothelial cells persist only transiently due to the insufficient recruitment of perivascular cells. We hypothesize that a combination of the two approaches may act synergistically to yield a better result. To enhance the recruitment of perivascular cells, human umbilical vein endothelial cells were genetically modified to overexpress platelet-derived growth factor (PDGF)-BB. PDGF-BB overexpression promoted both proliferation and migration of perivascular precursor cells (10T1/2 cells) in vitro. When mock-infected endothelial cells were implanted alone in vivo, they formed transient blood vascular networks that regressed by day 30. PDGF-BB overexpression enhanced the survival of endothelial cells in vivo. However, the PDGF-BB-expressing vessel network failed to establish patent blood flow. Co-implantation of PDGF-BB-overexpressing endothelial cells with 10T1/2 cells paradoxically resulted in the rapid regression of the vascular networks in vivo. PDGF-BB stimulated the expression of both chemokine (C-C motif) ligand 2 (CCL2) and CCL7 in 10T1/2 cells and led to the increased accumulation of macrophages in vivo. These results suggest a potential negative interaction between angiogenic growth factors and vascular cells; their use in combination should be carefully tested in vivo for such opposing effects. Angiogenesis, the growth of new blood vessels, is critical during tissue repair and regeneration.1Carmeliet P Angiogenesis in life, disease and medicine.Nature. 2005; 438: 932-936Crossref PubMed Scopus (2820) Google Scholar In cases of insufficient or impaired angiogenesis, the injured tissue remains dysfunctional and may suffer from irreversible damage.1Carmeliet P Angiogenesis in life, disease and medicine.Nature. 2005; 438: 932-936Crossref PubMed Scopus (2820) Google Scholar To combat this, two broad approaches have been used for enhancing angiogenesis: (i) delivery of pro-angiogenic genes (by gene transfer) or proteins (by bolus injection or controlled release devices) for endothelial and perivascular cell recruitment,2Jain RK Molecular regulation of vessel maturation.Nat Med. 2003; 9: 685-693Crossref PubMed Scopus (2043) Google Scholar and (ii) delivery of endothelial cells alone or together with perivascular cells.3Jain RK Au P Tam J Duda DG Fukumura D Engineering vascularized tissue.Nature Biotechnol. 2005; 23: 821-823Crossref Scopus (616) Google Scholar Therapeutic angiogenesis requires temporally and spatially orchestrated delivery of growth factors to form a functional and durable vasculature. A number of studies have shown that the use of a single angiogenic factor fail to form a mature and stable vasculature. For example, injection of an adenoviral vector expressing vascular endothelial growth factor (VEGF) into normal tissue leads to highly disorganized, leaky, and hemorrhagic vessels.4Pettersson A Nagy JA Brown LF Sundberg C Morgan E Jungles S Carter R Krieger JE Manseau EJ Harvey VS Eckelhoefer IA Feng D Dvorak AM Mulligan RC Dvorak HF Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor.Lab Invest. 2000; 80: 99-115Crossref PubMed Scopus (363) Google Scholar Furthermore, VEGF can potentiate inflammation by increasing the expression of adhesion molecules or the release of chemokines.5Detmar M Brown LF Schon MP Elicker BM Velasco P Richard L Fukumura D Monsky W Claffey KP Jain RK Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice.J Invest Dermatol. 1998; 111: 1-6Crossref PubMed Scopus (463) Google Scholar, 6Lee TH Avraham H Lee SH Avraham S Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells.J Biol Chem. 2002; 277: 10445-10451Crossref PubMed Scopus (154) Google Scholar, 7Heil M Clauss M Suzuki K Buschmann IR Willuweit A Fischer S Schaper W Vascular endothelial growth factor (VEGF) stimulates monocyte migration through endothelial monolayers via increased integrin expression.Eur J Cell Biol. 2000; 79: 850-857Crossref PubMed Scopus (100) Google Scholar, 8Melder RJ Koenig GC Witwer BP Safabakhsh N Munn LL Jain RK During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium.Nat Med. 1996; 2: 992-997Crossref PubMed Scopus (396) Google Scholar In addition, the resulting vessels are highly unstable and regress on removal of the angiogenic stimulus. Maturation and stabilization of new vessels requires perivascular cells.2Jain RK Molecular regulation of vessel maturation.Nat Med. 2003; 9: 685-693Crossref PubMed Scopus (2043) Google Scholar, 9Hirschi KK D'Amore PA Pericytes in the microvasculature.Cardiovasc Res. 1996; 32: 687-698Crossref PubMed Google Scholar Perivascular cells such as vascular smooth muscle cells and pericytes are thought to provide structural integrity to the blood vessels, lay down the extracellular matrix, and provide necessary survival factors to the endothelial cells.2Jain RK Molecular regulation of vessel maturation.Nat Med. 2003; 9: 685-693Crossref PubMed Scopus (2043) Google Scholar One potential way to enhance vessel maturation is by sequentially delivering VEGF and platelet-derived growth factor (PDGF)-BB.10Richardson TP Peters MC Ennett AB Mooney DJ Polymeric system for dual growth factor delivery.Nature Biotechnol. 2001; 19: 1029-1034Crossref Scopus (1567) Google Scholar Such a strategy was shown to induce mature vascular networks. However, the time required for host blood vessels infiltration by this strategy may limit the amount of ischemic tissue rescued. Another limitation with this type of therapeutic angiogenesis is that the neovascular response is reduced with age, and by underlying conditions such as hyperglycemia and atherosclerosis.11Simons M Angiogenesis: where do we stand now?.Circulation. 2005; 111: 1556-1566Crossref PubMed Scopus (362) Google Scholar Patients with diabetes mellitus or elevated homocysteine exhibit impaired angiogenic response.12Waltenberger J Impaired collateral vessel development in diabetes: potential cellular mechanisms and therapeutic implications.Cardiovasc Res. 2001; 49: 554-560Crossref PubMed Scopus (268) Google Scholar, 13Duan J Murohara T Ikeda H Sasaki K Shintani S Akita T Shimada T Imaizumi T Hyperhomocysteinemia impairs angiogenesis in response to hindlimb ischemia.Arterioscler Thromb Vasc Biol. 2000; 20: 2579-2585Crossref PubMed Scopus (79) Google Scholar Alternatively, vascular cells can be directly injected or implanted in the ischemic tissue. We have previously shown that human umbilical vein endothelial cells (HUVECs) implanted in a collagen gel in severe combined immunodeficient (SCID) mice form functional engineered vessels. However, the vessels were only transiently perfused, did not recruit adequate number of perivascular cells from the host tissue, and regressed within a few weeks. In contrast, co-implanting HUVECs with mouse perivascular cell precursors allowed pericyte investment and the formation of stable, durable, and functional engineered vessels.14Koike N Fukumura D Gralla O Au P Schechner JS Jain RK Tissue engineering: creation of long-lasting blood vessels.Nature. 2004; 428: 138-139Crossref PubMed Scopus (585) Google Scholar Thus, providing exogenous perivascular cells overcame the inability of the implanted endothelial cells to recruit host perivascular cells within a critical timeframe for the vessels to become mature and stabilized. Nevertheless, translation of this approach will require sources of both endothelial and perivascular cells in sufficient numbers for cell-based therapy of ischemic tissues. So far, such sources remain elusive, despite promising but preliminary results obtained with bone marrow-derived precursor cells or embryonic stem cells.15Dimmeler S Zeiher AM Schneider MD Unchain my heart: the scientific foundations of cardiac repair.J Clin Invest. 2005; 115: 572-583Crossref PubMed Scopus (596) Google Scholar, 16Wang ZZ Au P Chen T Shao Y Daheron LM Bai H Arzigian M Fukumura D Jain RK Scadden DT Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo.Nature Biotechnol. 2007; 25: 317-318Crossref Scopus (264) Google Scholar We hypothesized that a combination of cellular and molecular engineering approaches will act synergistically to yield the formation of a stable and durable network of blood vessels. To this end, we engineered endothelial cells to overexpress PDGF-BB, a mitogen and a chemoattractant for perivascular cells. We hypothesized that by increasing the levels of PDGF-BB expressed by endothelial cells, it may hasten the recruitment of perivascular cells from the host in an appropriate timeframe to stabilize the nascent blood vessels. HUVECs were obtained from Center of Excellence in Vascular Biology, Brigham and Women’s Hospital, Boston and maintained in Endothelial Cell Growth Medium (EGM) (Lonza, Basel, Switzerland). 293ET packaging cells were a kind gift from Dr. Brian Seed (Massachusetts General Hospital, Boston). 10T1/2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). All cells were maintained at 37°C in a humidified 5% CO2 incubator. The enhanced green florescent protein (EGFP) retrovirus vector, PBMN-I-EGFP was kindly provided by Dr. Gary Nolan (Stanford, CA). Human PDGF-B cDNA was purchased from InvivoGen (San Diego, CA). Full length PDGF-B cDNA was subcloned into the retroviral vector by first using PCR to add BamHI and NotI restriction sites with the following set of primers: forward primer-5′-GAATTCGGATCCATGAATCGCTGCTGGGCG-3′ and reverse primer-5′-AAGCTTGCGGCCGCCTAGGCTCCGAGGGTCTC-3′. The PCR product was cut with BamHI and NotI restriction enzymes and then inserted into the multicloning sites of PBMN-I-EGFP vector. For retrovirus packaging, the plasmids of PBMN-I-EGFP, Gag-pol, and VSVG (15 μg, 7 μg, and 5 μg, respectively) were mixed and co-transfected into 293ET cells with lipofectamine 2000 (Invitrogen, Carlsbad, CA) per manufacturer’s protocol. After overnight incubation, the 293ET cells were washed with PBS and then given Dulbecco’s Modified Eagle Medium 10% fetal bovine serum. The next day, the supernatant containing retrovirus was collected and fresh media was added; this step was repeated three more times. After the supernatant was collected, it was passaged through a 0.45 μm filter (Whatman, Brentford, UK) and was either used immediately for infection or kept at −80°C. For the transduction of HUVECs, the supernatant was first diluted 1:1 with fresh EGM and supplemented with polybrene (8 μg/ml). The diluted supernatant was then added to a subconfluent monolayer of HUVECs and allowed to incubate for 4 hours. Fresh EGM medium was exchanged at the end of the incubation period and this step was repeated 2 to 3 times on consecutive days. After 2 to 3 rounds of infection, more than 90% of HUVECs expressed the gene of interest as assessed by the expression of EGFP. HUVECs expressing EGFP only or co-expressing PDGF-BB and EGFP will be referred to as HUVEC-EGFP and HUVEC-PDGF-BB hereafter. HUVECs were grown in gelatin coated 10 cm dishes. After 4 days of culture, secreted and cell-retained fractions of PDGF-BB were quantified by enzyme-linked immunosorbent assay per manufacturer’s instruction (R&D Systems, Minneapolis, MN). For measuring secreted PDGF-BB, supernatant was collected and filtered through a 0.22-μm filter before measurement. For cell retained PDGF-BB, HUVECs were first washed with PBS, and then incubated with 1M/L solution of NaCl for 30 minutes on ice. The salt solution disrupts the charge–charge interaction between PDGF-BB and the extracellular matrix. The salt solution was then filtered with a 0.22-μm filter before measurement. For PDGF receptor (PDGFR) phosphorylation, 10T1/2 cells were starved overnight in 0.5% fetal bovine serum. The cells were then exposed to conditioned media from HUVEC-PDGF-BB for 5 minutes to stimulate the phosphorylation of PDGF receptor (PDGFR). The cells were washed three times with PBS containing 1 mmol/L NaVO4 and 50 mmol/L NaF and then were lysed with RIPA buffer containing protease inhibitor and phosphatase inhibitor. Immunoprecipitation was performed by the addition of 5 μl of anti-PDGFR-β antibodies (#3162) (Cell Signaling, Danvers, MA) to cell lysates containing 0.5 mg of total protein and was incubated overnight at 4°C. The next day, the antigen-antibody conjugate was precipitated with Agarose A/G. Phosphorylated PDGFR-β was immunoblotted with an anti-phosphotyrosine antibody conjugated with horseradish peroxidase (clone 4G10) (Upstate, Charlottesville, VA) and total PDGFR-β was immunoblotted with an anti-PDGFR-β antibody (#3162) (Cell Signaling). For VEGF receptor 2 (VEGFR-2) phosphorylation, EGFP-HUVEC and PDGF-BB-HUVEC cells were incubated in serum-free medium for 1 hour and then incubated with or without 50 ng/ml VEGF for 2 to 5 minutes. Then the cells were scraped from plates, pelleted, and resuspended in lysis buffer; 60 μg of protein per sample was separated on a 4% to 15% acrylamide gradient gel (Bio-Rad, Hercules, CA). The expression of phopho-VEGFR2, VEGFR2, and actin were detected by polyclonal antibodies against VEGFR2 (1:1000) and phosphorylated VEGFR2 (1:2000) (Cell Signaling), and by monoclonal antibodies against actin (1:5000) (Sigma). The activity of the ectopically expressed soluble PDGF-BB in HUVECs was assayed by testing the ability of cell culture supernatant to induce the proliferation of 10T1/2 cells. Briefly, confluent monolayers of HUVECs that had been transduced with PDGF-BB or EGFP were incubated for 12 hours with Dulbecco’s Modified Eagle Medium containing 1% fetal bovine serum. The conditioned medium was filtered with a 0.22 μm filter and stored in −80°C until ready for use. 10T1/2 cells were plated in a 96 well plate at a density of 5000 cells/well. The next day, the medium in each well was removed and replaced with 100 μl of HUVECs conditioned medium and allowed to incubate for 24 hours. After 24 hours, 20 μl of 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (20 μg/ml, Sigma-Aldrich, St. Louis, MO) was added to each well and the plate was incubated further for 2 hours. The medium was then removed and replaced with 100 μl of dimethyl sulfoxide. The absorbance was read with a colorimetric plate reader at 550 to 655 μm wavelength. Cell migration was assessed using Falcon HTS FluoroBlok 24-well inserts (BD Biosciences, San Jose, CA) with 3-μm pores. EGFP-10T1/2 cells (2 × 104) suspended in 250-μl Basal Medium Eagle (Invitrogen) with 0.5% fetal bovine serum were placed inside each insert and 5 × 104 per well HUVECs suspended in 800 μl EGM (Lonza) were plated on a 24-well plate. The cells were kept overnight. The next day, the cell culture media of both HUVECs and 10T1/2 cells were changed to Basal Medium Eagle with 0.5% fetal bovine serum, and then the inserts were placed in the respective wells. At 4, 8, and 12 hours, the bottom of each insert was imaged in fluorescence using an inverted fluorescence microscope (Olympus IX70, Center Valley, PA) equipped with a motorized stage and motorized filter wheel (Improvision Inc, Lexington, MA). Transmigrated cells were quantified using ImageJ (http://rsb.info.nih.gov/ij/) by using threshold function and measuring the area covered by the migrated cells. In vitro tube formation was studied using previously described procedures.16Wang ZZ Au P Chen T Shao Y Daheron LM Bai H Arzigian M Fukumura D Jain RK Scadden DT Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo.Nature Biotechnol. 2007; 25: 317-318Crossref Scopus (264) Google Scholar Matrigel (Collaborative Biomedical Products, Bedford, MA) was diluted with Endothelial Cell Growth Medium in 1:1 ratio. Next, 60 μl of the solution were added to each well of a 96-well plate and allowed to form a gel at 37°C for 30 minutes. EGFP-HUVEC and PDGFBB-HUVEC (20,000 cells) cells in 200 μl of complete medium were subsequently added to each well and incubated overnight at 37°C in 5% CO2. Under these conditions, both EGFP- and PDGFBB-HUVEC cells formed delicate networks of tubes that were fully developed after 16 hours. In situ TdT-mediated dUTP nick end labeling (ApopTag peroxidase In situ detection kit, Chemicon, Temecula, CA) was used according to manufacturer’s instructions to identify apoptotic cells. EGFP-HUVEC and PDGFBB-HUVEC cells were cultured in serum-free medium, in complete medium, or under hypoxic condition (1% O2 to 5% CO2 balance N2 was used). One million endothelial cells and 2 × 105 10T1/2 cells were suspended in 1 ml solution of rat-tail type 1 collagen (1.5 mg/ml) (BD Biosciences) and human plasma fibronectin (90 μg/ml) (Sigma) in 25 mmol/L Hepes (Sigma) buffered EGM medium at 4°C. The pH was adjusted to 7.4 by using 1N NaOH (Fisher Science, City, NJ). The cell suspension was pipetted into 12-well plates and warmed to 37°C for 30 minutes to allow polymerization of collagen. Each solidified gel construct was covered by 1 ml of warmed EGM medium. After 1 day culture in 5% CO2, a skin puncher was applied to create circular disk-shape pieces of the construct (4-mm diameter), and they were implanted into the cranial windows in SCID mice.14Koike N Fukumura D Gralla O Au P Schechner JS Jain RK Tissue engineering: creation of long-lasting blood vessels.Nature. 2004; 428: 138-139Crossref PubMed Scopus (585) Google Scholar, 17Yuan F Salehi HA Boucher Y Vasthare US Tuma RF Jain RK Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows.Cancer Res. 1994; 54: 4564-4568PubMed Google Scholar Multiphoton laser-scanning microscopy was used to visualize and quantify the morphological changes in EGFP-expressing HUVECs. The perfused vessels were highlighted by tail vein injection of 1% tetramethylrhodamine-labeled dextran (MW 2,000,000), indicating the formation of functional engineered vessels14Koike N Fukumura D Gralla O Au P Schechner JS Jain RK Tissue engineering: creation of long-lasting blood vessels.Nature. 2004; 428: 138-139Crossref PubMed Scopus (585) Google Scholar, 17Yuan F Salehi HA Boucher Y Vasthare US Tuma RF Jain RK Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows.Cancer Res. 1994; 54: 4564-4568PubMed Google Scholar The cord formation assay was performed identically as above except that the tissue engineered construct was allowed to culture in vitro. Images of the tissue-engineered construct were taken randomly at 3, 7, and 12 days with an inverted fluorescence microscope. The formation of vessel-like network was quantified by thresholding the image and measuring the area of vessel-like structures with Image J software. 10T1/2 cells were serum starved overnight and stimulated with 50 ng/ml of recombinant human PDGF-BB (R&D Systems) for 2 or 4 hours. Total RNA was isolated from 10T1/2 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quantity and purity of RNA were determined by UV absorbance at 260 and 280 nm. For PCR array analysis, cDNA was first synthesized with RT2 First Strand Kit Array (SABiosciences, Frederick, MD) and the samples were then analyzed with Mouse Inflammatory Cytokines & Receptors RT2 Profiler PCR Array (SABiosciences). All procedures were performed according to manufacturer’s instructions. For real-time quantitative PCR, cDNA was synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). qRT-PCR was performed using the 7300 Real-Time PCR System and Power SYBR Green PCR Master Mix (Applied Biosystems). Primers were designed using Primer Express (Applied Biosystems) and purchased from Integrated DNA Technologies (Coralville, IA). Primer specificity for each gene of interest was confirmed by comparison with known sequences in the BLAST database (National Center for Biotechnology Information). Samples were analyzed in triplicates, and the gene expression level for each sample was normalized to the corresponding glyceraldehyde-3-phosphate dehydrogenase expression level, to control for loading differences. Negative controls were performed for each sample using non-reverse-transcribed RNA. HUVECs were transduced with retroviral constructs to stably overexpress either PDGF-BB and EGFP or EGFP alone (Figure 1, A and B). The expression of PDGF-BB was quantified by enzyme-linked immunosorbent assay in the transduced HUVECs (Figure 1, C and D). In the case of HUVECs overexpressing PDGF-BB, we observed a trend of a higher ratio of soluble to cell-associated fraction of PDGF-BB, possibly due to saturation of binding sites on the cell surface (Figure 1E). Alternatively, the amount of cell-associated PDGF-BB might be underestimated since majority of PDGF-BB is secreted basally in endothelial cells.18Zerwes HG Risau W Polarized secretion of a platelet-derived growth factor-like chemotactic factor by endothelial cells in vitro.J Cell Biol. 1987; 105: 2037-2041Crossref PubMed Scopus (60) Google Scholar Overexpression of PDGF-BB did not significantly change the phenotype of the endothelial cells (ie, migration rate, VEGFR2 signaling, tube formation) (see supplemental Figures S1 and S2 at http://ajp.amjpathol.org). However, PDGF-BB overexpression reduced the proliferation rate of HUVECs (see supplemental Figure S1a at http://ajp.amjpathol.org). Next, we examined the paracrine effects of PDGF-BB overexpression in endothelial cells on 10T1/2 cells, a line of mouse embryonic fibroblast that mimics the behavior of pericyte. HUVEC-PDGF-BB-conditioned medium stimulated the phosphorylation of the PDGF-Rβ in 10T1/2 cells and enhanced cell proliferation compared with control medium (Figure 2, a and b). Using Boyden chamber migration assay, we found that overexpression of PDGF-BB in HUVECs promoted the migration of 10T1/2 cells in vitro(Figure 2c). These findings demonstrated that the PDGF-BB overexpressed in HUVECs was functionally active on 10T1/2 perivascular cell precursors in vitro. To create engineered vessels, we implanted HUVEC-PDGF-BB cells in a fibronectin/collagen matrix onto the pial surface of the brain in a cranial window in SCID mice.14Koike N Fukumura D Gralla O Au P Schechner JS Jain RK Tissue engineering: creation of long-lasting blood vessels.Nature. 2004; 428: 138-139Crossref PubMed Scopus (585) Google Scholar For control, we implanted HUVEC expressing EGFP alone. We tracked the cells in vivo with multiphoton laser scanning microscopy and monitored the kinetics of blood vessel formation. In the group implanted with HUVEC-EGFP cells, the endothelial cells became elongated and interconnected, forming a mesh-like network on day 3 (Figure 3A). Multiple vacuoles were observed to have formed within the endothelial cells. By day 7, some of these vacuoles had coalesced into luminal structure (Figure 3A). On day 18, blood flow was seen within the lumen of the endothelium suggesting that the engineered blood vessels had formed functional connection to the host circulatory network (Figure 3A). Similar to previous results,14Koike N Fukumura D Gralla O Au P Schechner JS Jain RK Tissue engineering: creation of long-lasting blood vessels.Nature. 2004; 428: 138-139Crossref PubMed Scopus (585) Google Scholar engineered blood vessels derived from HUVEC only were unstable and most of the vessels had regressed by day 30 (Figure 3, A and C). In contrast, in animals implanted with HUVEC-PDGF-BB, the endothelial cells persisted beyond 30 days. Similar to the control group, HUVEC-PDGF-BB formed a mesh-like network with lumen inside it. However, few of these vascular structures were perfused with blood at day 30 (Figure 3, B and D). Next, we co-implanted HUVECs with 10T1/2 cells to determine whether exogenously supplied perivascular cell precursors could enhance the formation of functional engineered blood vessels and enhanced the anastamosis of HUVEC-PDGF-BB-derived vessels to the host circulation. In the control group with HUVEC-EGFP and 10T1/2 cells, HUVEC-EGFP formed a vessel-like network at day 4. By day 8, blood flow was detectable inside the newly formed lumens (Figure 4A). Nevertheless, we found that co-implantation of HUVEC-PDGF-BB and 10T1/2 cells led to an accelerated reduction in the density of HUVECs in vivo compared with the control (Figure 4B). Quantification of the total vessel length density showed a reduction in the number of HUVEC-derived vessels at day 4, and the density dropped precipitously thereafter (Figure 4, C and D). Next, we sought to determine whether this unexpected in vivo phenotype was due directly to the presence of 10T1/2 cells or indirectly through a change in the in vivo microenvironment. HUVEC-PDGF-BB and HUVEC-EGFP were incorporated with 10T1/2 cells in fibronectin/collagen matrix and cultured in vitro. After 3 days, we observed the formation of vessel-like structures by the HUVECs similar to those formed in vivo(Figure 5, A and B). There was no significant difference in the ability of HUVEC-PDGF-BB and HUVEC-EGFP to morph into the vessel-like structures. The vessel-like structures in both groups regressed at similar rate with the progression of time and by day 12, only a few of these structures remained in the collagen gel (Figure 5C). These data suggest HUVEC-PDGF-BB and HUVEC-EGFP—when co-cultured with 10T1/2 cells—morphed into vessel-like structures with similar efficiency in vitro. The inability of co-implanted HUVEC-PDGF-BB and 10T1/2 cells to form stable blood vessels in vivo is likely secondary to in vivo alterations of the local microenvironment by the overexpression of PDGF-BB. We next tested the new hypothesis that the PDGF-BB-induced change in microenvironment caused the regression of the implanted endothelial cells. We implanted two groups of mice with genetically modified endothelial cells. In the experimental group, we co-implanted HUVEC-PDGF-BB, HUVEC-DsRed, and 10T1/2 cells. In the control group, we co-implanted HUVEC-EGFP, HUVEC-DsRed, and 10T1/2 cells. In the control group, we observed that both HUVEC-EGFP and HUVEC-DsRed formed vessel-like network and persisted for more than 11 days (Figure 6). In contrast, HUVEC-DsRed co-implanted with HUVEC-PDGF-BB and 10T1/2 cells showed a much smaller number of cells surviving at day 11. This result suggests that 10T1/2 cells stimulated by PDGF-BB caused the regression of the implanted endothelial cells irrespective of their specific genetic modification. To examine the effects of PDGF-BB stimulation on 10T1/2 cells, we performed PCR array analysis on 10T1/2 cells with or without PDGF-BB stimulation (see supplementary Table S1 at http://ajp.amjpathol.org). Initial screening revealed that CCL2 (MCP-1), CCL7 (MCP-3), and Spp1 were up-regulated. We confirmed the up-regulation of CCL2 and CCL7 by quantitative real-time PCR (Figure 7, A and B). Since both CCL2 and CCL7 are critical chemokines for monocyte/macrophage recruitment, we performed immunohistochemistry for infiltration of inflammatory cells. We found a significantly higher number of F4/80 positive macrophages in animals implanted with HUVEC-PDGF-BB and 10T1/2 cells (Figure 7, C–E). This finding suggests that PDGF-BB overexpression leads to an increased in local inflammation. Therapeutic angiogenesis has great promise to alleviate tissue ischemia and to help repair damaged tissues. Presently, a number of clinical trials are testing different angiogenic agents, such as VEGF and basic fibroblast growth factor, to induce new blood vessels in poorly perfused tissue with the ultimate goal of improved tissue and organ function.11Simons M Angiogenesis: where do we stand now?.Circulation. 2005; 111: 1556-1566Crossref PubMed Scopus (362) Google Scholar In parallel, cell-based therapy with bone marrow-derived progenitor and/or embryonic stem cells is also being investigated for regener" @default.
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• W2081167583 date "2009-07-01" @default.
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• W2081167583 title "Paradoxical Effects of PDGF-BB Overexpression in Endothelial Cells on Engineered Blood Vessels In Vivo" @default.
• W2081167583 cites W1965621019 @default.
• W2081167583 cites W1973358829 @default.
• W2081167583 cites W1974171881 @default.
• W2081167583 cites W1984803858 @default.
• W2081167583 cites W1990793260 @default.
• W2081167583 cites W1994778563 @default.
• W2081167583 cites W2002495134 @default.
• W2081167583 cites W2003643174 @default.
• W2081167583 cites W2013297672 @default.
• W2081167583 cites W2026349111 @default.
• W2081167583 cites W2035371785 @default.
• W2081167583 cites W2047470479 @default.
• W2081167583 cites W2049185799 @default.
• W2081167583 cites W2052398489 @default.
• W2081167583 cites W2078481843 @default.
• W2081167583 cites W2083183739 @default.
• W2081167583 cites W2089004231 @default.
• W2081167583 cites W2098799479 @default.
• W2081167583 cites W2103671501 @default.
• W2081167583 cites W2105028749 @default.
• W2081167583 cites W2114834794 @default.
• W2081167583 cites W2116008315 @default.
• W2081167583 cites W2121822802 @default.
• W2081167583 cites W2140227503 @default.
• W2081167583 cites W2142084529 @default.
• W2081167583 cites W2144026946 @default.
• W2081167583 cites W2156606731 @default.
• W2081167583 cites W2163926528 @default.
• W2081167583 cites W2167060652 @default.
• W2081167583 cites W304295984 @default.
• W2081167583 cites W4242965605 @default.
• W2081167583 cites W4251787912 @default.
• W2081167583 doi "https://doi.org/10.2353/ajpath.2009.080887" @default.
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