HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Free All Versions of this Article: 95/5/2081    most recent 00232.2003v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (49) Citing Articles via Google Scholar Google Scholar Articles by Yamamoto, K. Articles by Ando, J. Search for Related Content PubMed PubMed Citation Articles by Yamamoto, K. Articles by Ando, J.

Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress

¹Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 113-0033 Tokyo; ²Department of Internal Medicine, Tokyo Medical University, 160-8402 Tokyo; ³Department of Physiology, School of Medicine, Tokai University, 259-1193 Isehara; and ⁴Interdisciplinary Science Center, Nihon University, 102-0074 Tokyo, Japan

Submitted 5 March 2003 ; accepted in final form 1 July 2003

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Endothelial progenitor cells (EPCs), circulating in peripheral blood, migrate toward target tissue, differentiate, and contribute to the formation of new vessels. In this study, we report that shear stress generated by blood flow or tissue fluid flow can accelerate the proliferation, differentiation, and capillary-like tube formation of EPCs. When EPCs cultured from human peripheral blood were subjected to laminar shear stress, the cells elongated and oriented their long axes in the direction of flow. The cell density of the EPCs exposed to shear stress was higher, and a larger percentage of these cells were in the G₂-M phase of the cell cycle, compared with EPCs cultured under static conditions. Shear stress markedly increased the EPC expression of two vascular endothelial growth factor receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase-1, and an intercellular adhesion molecule, vascular endothelial-cadherin, at both the protein and mRNA levels. Assays for tube formation in the collagen gels showed that the shear-stressed EPCs formed tubelike structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that EPCs are sensitive to shear stress and that their vasculogenic activities may be modulated by shear stress.

blood vessels; angiogenesis; neovascularization; mechanical stress; vascular endothelial growth factor

During the process of EPC incorporation into tissues and neovascularization, the cells are exposed to fluid shear stress, a mechanical force generated by blood flow or interstitial fluid flow (42). Ample evidence has shown that shear stress modulates mature EC function and gene expression, playing important roles in the homeostasis of vascular functions and the mechanisms of blood flow-dependent phenomena, such as angiogenesis, vascular remodeling, and atherogenesis (12). Accordingly, the present study was designed to examine the effects of shear stress on EPCs. Using a flow-loading apparatus, we applied controlled levels of laminar shear stress to EPCs isolated from human peripheral blood and examined the changes in their morphology, proliferation, and expression of EC-specific marker proteins. We also examined the effect of shear stress on tube formation by EPCs in collagen gels.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Mononuclear cell isolation and culture. Mononuclear cells were isolated from 100 ml of human peripheral blood by density gradient centrifugation by using Vacutainer CPT tubes (Becton Dickinson) containing Histopaque-1077 (Sigma), as previously described (6). Cells were then plated on culture dishes coated with 100 µg/ml of human fibronectin (Sigma) at a density of 5 x 10⁵ cells/cm² and cultured in EBM-2 (Clonetics) supplemented with 5% fetal bovine serum, human VEGF, human FGF-2, human epidermal growth factor, insulin-like growth factor-1, and ascorbic acid at 37°C in an atmosphere of 5% CO₂. Four days after seeding, 10% of the cells had attached to the dishes, whereas the remaining 90% were nonadherent. Nonadherent cells were removed by washing with PBS, and the media were reapplied. The culture was maintained through days 4-21.

Identification of EPCs. To confirm the EPC phenotype, attached mononuclear cells were incubated with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (DiI-acLDL; 10 µg/ml; Biomedical Technologies) at 37°C for 1 h. The cells were then fixed with 2% paraformaldehyde for 10 min and incubated with FITC-labeled Ulex europeus agglutinin (lectin, 10 µg/ml; Sigma) for 1 h. After staining, the samples were viewed with an inverted fluorescent microscope (Nikon). Cells demonstrating double-positive fluorescence were identified as differentiating EPCs (7, 19, 20). Nearly all adherent cells (>95%) were DiI-acLDL(+) Ulex-lectin(+).

Shear stress experiments. Cells were exposed to laminar shear stress by using a rotating disk-type flow loading device, as previously described (4, 32). Briefly, a dish containing cultured EPCs was placed on the stage of the device, and a stainless steel disk was placed in the bottom of the dish. The rotation of the disk caused the media to flow in a concentric circle, thereby exerting a laminar shear stress on the cells. The intensity of the shear stress () was calculated as = µ·r/d, where µ is the fluid viscosity, r is the distance from the center of the dish, is the angular velocity, and d is the distance between the disk and the dish. The device generates gradients of shear stress that are dependent on the distance from the center of the dish. In the majority of experiments, cells from a single dish were used, and the shear stress values were averaged. In some experiments, bands of cells at different radii were separately scraped from the same dish and studied. The shear stress used in this study ranged from 0.1 to 2.5 dyn/cm²; all experiments were performed at 37°Cin a CO₂ incubator.

Flow cytometry. The expression of EC-specific marker proteins was measured by using a FACScan flow cytometer (Becton Dickinson). A total of 2 x 10⁵ cells were incubated with monoclonal antibodies against VEGF receptors, such as kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase-1 (Flt-1) (Sigma), and adhesion molecules, such as vascular endothelial (VE)-cadherin (RDI), for 45 min at 4°C. The cells were then incubated with FITC-conjugated anti-mouse IgG (H+L) antibody (Amersham) for 30 min at 4°C and analyzed by FACS. The accumulated cells were segregated into monocyte-sized and lymphocyte-sized fractions by gating during a light-scatter analysis. Monocyte-sized, but not lymphocyte-sized, cells were positive for lectin and DiI-acLDL, whereas lymphocyte-sized, but not monocyte-sized, cells were positive for leukocyte common antigen CD45 (DAKO). Thus the gating of the monocyte-sized fraction was programmed to ensure the accumulation of EPCs. Cell number vs. logarithmic fluorescence intensity histograms were recorded for 20,000 cells per sample. Background fluorescence was obtained from the negative control cells stained with FITC-conjugated anti-mouse IgG (H+L) antibody and subtracted from the mean channel fluorescence of the specific staining patterns. The expression of each antigen was expressed as the mean channel fluorescence.

Cell cycle analysis. Cells were detached from dishes by incubating in PBS supplemented with 2 mM EDTA. The detached cells were then washed with PBS and fixed with 70% ethanol for 1 h at 4°C. After washing with PBS, the cells were incubated with PBS containing 0.5% RNase (Sigma) for 30 min at room temperature and then stained with propidium iodide (50 µg/ml) for 15 min. The fluorescence intensity of this dye was measured in a sample containing 20,000 cells, and the cell cycle was analyzed by using ModFit LT Cell Cycle Analysis Software (Verity Software House).

RT-PCR aalysis. RT-PCR was performed to determine the EC-specific marker mRNA levels, as previously described (23). Briefly, the reverse transcription of RNA was performed in a reaction mixture containing total RNA, moloney murine leukemia virus reverse transcriptase (GIBCO), oligo(dT)_12-18, ribonuclease inhibitor, each dNTP mixture, and dithiothreitol in a first-strand buffer. The mixture was incubated at 37°C for 1 h, heated to 99°C for 5 min, and then chilled at 4°C for 5 min. The cDNA samples were then coamplified by PCR by using the following primer pairs: KDR forward: 5'-AGACCAAAGGGGCACGATTC-3', reverse: 5'-CAGCAAAACACCAAAAGACCAGAC-3'; Flt-1 forward: 5'-ATTTGTGAT TTTGGCCTTGC-3', reverse: 5'-CAGGCTCATGAACTTGAAAGC-3'; VE-cadherin forward: 5'-GCTGAAGGAAAACCAGAAGAAGC-3', reverse: 5'-TCGTGATTATCCGTG AGGGTAAAG-3'; GAPDH forward: 5'-ACATCATCCCTGCCTCTACTGG -3', reverse: 5'-AGTGGGTGTCGCTGTTGAAGTC-3'. A solution containing ExTaq DNA polymerase (Takara), [-³²P]dCTP, and each primer was added to each sample. Each temperature cycle consisted of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The amplified product was sampled every other cycle and electrophoresed on a 5% polyacrylamide gel. The PCR product sequences confirmed that each mRNA product had been correctly amplified by its primers.

For the quantification of PCR products, the radioactivity of each band was measured by using a GS363 Molecular Imager System (Bio-Rad) and plotted against the number of PCR cycles on a semilogarithmic scale, forming a sigmoid curve. From the curve, the cycle in which the operating range of the PCR was linear was selected, and the ratio of radioactivity between each target gene and GAPDH in the cycle was calculated as a parameter of relative mRNA levels.

Tube formation assay. Collagen gels were formed by adding Biocoat Matrigel (Becton Dickinson) into 24-well plates and incubating in a CO₂-free incubator at 37°C for 30 min. The same batch of Matrigel was used for all of the experiments. The gels were then overlaid with 1 x 10⁵ cells suspended in culture medium and incubated at 37°C in an atmosphere of 5% CO₂. Gels were examined by using a phase-contrast microscope equipped with a digital camera PDMC (Polaroid); the images were then imported as TIFF files into National Institutes of Health (NIH) Image software. A second observer measured the total length of the tubelike structures (defined as those exceeding 200 µm in length) per each image.

Statistical analysis. All results are expressed as means ± SD. The statistical significance was evaluated by using an ANOVA and a Bonferroni adjustment applied to the results of a t-test using SPSS (SPSS). A value of P < 0.01 was regarded as being statistically significant.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
EPCs exhibit morphological changes in response to shear stress. EPCs attached, spread, and proliferated on tissue culture plastic coated with fibronectin and showed positive staining for both lectin and DiI-acLDL (Fig. 1, A-C). The EPCs showed no definite orientation under static conditions. When they were exposed to laminar shear stress for 24 h, they became elongated and oriented their long axes in the direction of flow (Fig. 1D). The elongated cells also showed positive staining for both lectin and DiI-acLDL (Fig. 1, E and F). When shear-stressed cells were returned to the static conditions, the changes reversed in 24 h (data not shown), indicating the morphological response to shear stress to be reversible.

View larger version (79K): [in this window] [in a new window]   Fig. 1. Effect of shear stress on endothelial progenitor cell (EPC) morphology. A and D: phase-contrast photomicrographs of EPCs cultured under static conditions (Static) or exposed to shear stress (Flow, 0.1-2.5 dyn/cm2 for 24 h). The cells shown were exposed to 2 dyn/cm2, which was calculated based on the distance of cells from the center of the dish. The EPCs became elongated and aligned in response to the shear stress. B and E: fluorescence images of lectin. C and F: fluorescence images of DiI-acLDL. All cells showed positive staining for both lectin and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (DiI-acLDL). Data from the day 10 culture are shown, but similar responses were obtained at other times on culture days 4 and 14. The direction of flow is left to right. The bar indicates 50 µm.

Shear stress augments EPC proliferation. EPCs were exposed to laminar shear stress for 24 h, and changes in cell density were determined on days 4, 6, 8, 10, and 14 (Fig. 2). Under static conditions, cell density increased with time but plateaued on day 10. Shear stress induced a much larger increase in cell density than occurred in static cells. Statistically significant differences were observed on days 8, 10, and 14 (P < 0.001).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Cell density as a function of time for control (Static) or shear-stressed (Flow, 0.1-2.5 dyn/cm2) EPCs. Cells were exposed to shear stress for 24 h on culture days (D) 3, 5, 7, 9, and 13, and the cell density (no. of cells positive for both lectin and DiI-acLDL per unit area) was measured by using photomicrographs obtained on D4, D6, D8, D10, and D14. Values are means ± SD for 5 separate samples. *P < 0.001 vs. static control.

Cell cycle analysis showed that the shear stress decreased the percentage of EPCs in the G₀ and G₁ phases of the cell cycle and increased the percentage of EPCs in the S, G₂, and M phases (Table 1).

These results indicate that laminar shear stress stimulates EPC proliferation.

Shear stress modulates expression of EC-specific markers in EPCs. The cell surface expression patterns of various EC-specific markers were examined in EPCs that had been either cultured under static conditions or subjected to shear stress. Flow cytometric analysis showed a time-dependent increase in the expressions of KDR, Flt-1, and VE-cadherin in EPCs cultured under static conditions (static; Fig. 3). The levels of expression of KDR, Flt-1, and VE-cadherin increased by about six-, five-, and fivefold, respectively, on day 21, compared with the values on day 4.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effect of shear stress on the expression of endothelial cell (EC)-specific marker proteins. EPCs cultured either under static conditions (Static) or exposed to shear stress (Flow, 0.1-2.5 dyn/cm2) were labeled with fluorescent antibodies to kinase insert domain-containing receptor (KDR; A), fms-like tyrosine kinase-1 (Flt-1; B), and vascular endothelial (VE)-cadherin (C), and the fluorescence intensity of 20,000-cell samples was measured by FACS. Cells were exposed to shear stress for 24 h on D5, D7, D9, D13, and D20, and assayed on D6, D8, D10, D14, and D21. D4, static control on culture day 4. Shear stress accelerated increases in the expression of KDR, Flt-1, and VE-cadherin. Values are means ± SD of 4 separate samples. Similar results were obtained in 3 separate experiments. *P < 0.01 vs. static control.

Shear stress modulated the expression of these EC-specific markers in EPCs. Shear stress markedly increased the levels of expression of KDR, Flt-1, and VE-cadherin (flow; Fig. 3). The levels of KDR, Flt-1, and VE-cadherin expression on days 8, 10, and 14 in the EPCs exposed to shear stress were almost identical to those on day 21 in the EPCs cultured under static conditions.

The mRNA levels of KDR, Flt-1, and VE-cadherin were determined by using RT-PCR. Shear stress markedly increased the KDR, Flt-1, and VE-cadherin mRNA levels in a magnitude-dependent manner at day 6 (Fig. 4). These results indicate that shear stress accelerates the expression of EC-specific markers on EPCs at both the protein and mRNA levels.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effect of shear stress on mRNA levels of EC-specific markers. A: KDR. B: Flt-1. C: VE-cadherin. Total RNA was extracted on D4 and D6 from EPCs that had been either incubated under static conditions (D4 static, D6 static) or exposed to shear stress for 24 h (D6 Flow-L, D6 Flow-H). Bands of cells at the inner or outer half of the same dish (Flow-L, 0.1-1.3 dyn/cm2; Flow-H, 1.4-2.5 dyn/cm2) were separately scraped out and analyzed. After reverse transcription of the RNA into cDNA, the cDNA was amplified by PCR using specific primers and electrophoresed on a polyacrylamide gel. Top: bands show the amplification products for each EC-specific marker and GAPDH mRNA. Bottom: bar graphs (means ± SD) represent the percent changes relative to static controls (D4 static), in band density normalized to GAPDH mRNA levels. Shear stress dose-dependently increased KDR, Flt-1, and VE-cadherin mRNA levels. Similar results were obtained for at least 3 separate experiments. *P < 0.01 vs. static control.

Shear stress promotes tube formation by EPCs in collagen gels. To investigate whether shear stress affects the ability of EPCs to form capillary-like tubes, EPCs cultured under static conditions and EPCs exposed to shear stress for 24 h were seeded in collagen gels and examined for tube formation microscopically (Fig. 5A). Control EPCs cultured under static conditions were present in the form of individual cells or small cellular aggregates at 2 h and exhibited clustering on day 1. Tubelike structures were observed on day 4. By contrast, shear-stressed EPCs appeared to be spindle shaped, elongated, and interconnected and had formed tightly adherent cords of cells as early as 2 h after seeding. Formation of tubelike structures became more prominent on day 1, and an extensive tubular network was observed on day 4. Quantitative analysis with NIH Image showed the total length of the tubes at 2 h and on days 1, 4, and 7 to be significantly greater in shear-stressed EPCs than in static control EPCs (Fig. 5B). These findings suggest that shear stress enhances the ability of EPCs to form tubelike structures in collagen gels.

View larger version (82K): [in this window] [in a new window]   Fig. 5. Tube formation in collagen gel. A: representative images captured after incubation for 2 h, and 1, 4, and 7 days. On culture day 6, cells that had been incubated under static conditions (Static) or exposed to shear stress (Flow, 0.1-2.5 dyn/cm2 for 24 h) were seeded onto collagen gels formed with Biocoat Matrigel. Tubelike structures appeared at 4 days in the static EPC cultures but were observed after 1 day in shear-stressed cells and became more extensive with time. The bar indicates 200 µm. B: quantification of tube formation. NIH Image was used to determine the total length of the tubelike structures in images captured after incubation for 2 h and 1, 4, and 7 days. Preexposure to shear stress clearly enhanced tubelike structure formation by EPCs. Values are means ± SD of 30 images from 3 separate experiments. *P < 0.001 vs. static control.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The present study demonstrated EPCs to be sensitive to shear stress, i.e., that EPCs change morphologically and functionally in response to shear stress. EPCs elongated and oriented their long axis in the direction of flow when exposed to shear stress (0.1-2.5 dyn/cm²) for 24 h. These morphological changes were observed in mature ECs, such as bovine aortic ECs and human umbilical vein ECs (13, 14). Mature ECs, however, align in the direction of flow in response to shear stress of >8-10 dyn/cm², but not to such low shear stress as 0.1-2.5 dyn/cm² (24). This indicates that, in terms of morphological responses to shear stress, EPCs are more sensitive than mature ECs.

Shear stress stimulated EPC proliferation. The effect of shear stress on mature EC proliferation, however, remains controversial. Laminar shear stress has been shown to inhibit DNA synthesis in human umbilical vein endothelial cells (HUVECs) in a confluent monolayer (1, 25), but to stimulate DNA synthesis in bovine aortic ECs exposed to shear stress during regeneration after monolayer scraping (3). On the other hand, animal experiments using arteriovenous shunts have shown elevated shear stress to induce an increase in EC proliferation in arteries (28). In the present study, the EPCs did not reach confluence during the 21-day culture period. Thus the absence of cell growth contact inhibition may be related to the behavior difference between EPCs and confluent HUVECs. In this sense, EPCs may not assume a complete endothelial phenotype (at least, not within 21 days). Shear stress may also cause non-EPCs to slough off, causing the EPCs to lose cell contact signals that inhibit proliferation and/or differentiation.

Expression of the VEGF receptors KDR and Flt-1 in EPCs gradually increased with the duration of cell culture. Shear stress markedly augmented the increases in KDR and Flt-1 expression in EPCs. These findings indicate that cells exposed to shear stress acquire enhanced responsiveness to endogenous VEGF. These cells may exhibit greater participation in ongoing angiogenesis. Shear stress has also been shown to cause tyrosine phosphorylation in KDR, without the participation of its ligand VEGF, and to trigger downstream signal transduction, including activation of ras and ERK (11). This means that shear stress activates KDR in the same manner as chemical stimulation with VEGF. Thus activation of KDR, as well as the increase in its expression, may be involved in the mechanism by which shear stress stimulates EPC proliferation.

VE-cadherin is present at endothelial adherens junctions and plays an important role in intercellular adhesion and cell differentiation, growth, and migration. Shear stress increased VE-cadherin expression in EPCs at both the protein and the mRNA level. Similar results were obtained in mature ECs (33). VE-cadherin has also been implicated in vasculogenesis and angiogenesis. A targeted null mutation in the VE-cadherin gene prevented the organization of vascular-like structures in mouse embryos (41). A targeted deficiency of the VE-cadherin gene in mice was also found to increase EC apoptosis and impair vascular remodeling and maturation, causing death on day 9.5 of gestation (9). In an in vitro model in which HUVECs were induced to form capillary-like tubes in fibrin and collagen gels, a monoclonal antibody against VE-cadherin inhibited the formation of capillary tubes, and, when the antibody was added to preformed capillary tubes, it disrupted the capillary network (8). The increase in VE-cadherin induced by shear stress may, therefore, contribute to tube formation and thereby lead to neovascularization.

Vascular ECs can organize into tubular capillary-like structures. An in vitro model in which ECs are cultured in three-dimensional collagen gels in the presence of basic FGF and VEGF has been widely used to study the processes involved in the formation of capillary tubes (43). Use of this model revealed that preconditioning EPCs with shear stress enhanced tube formation. EPCs were exposed to shear stress for 24 h on culture day 6 and assayed up to 1 wk later. The positive effect of shear stress on tube formation persisted for an entire week. There may be a "memory" effect of 24-h exposure to shear stress that lasts for, at least, 1 wk. The enhanced tube formation appears to be related to shear-stress-induced augmentation of cell growth, VEGF receptor expression, and VE-cadherin expression, although their exact relationships were not assessed in the present study.

In the present study, peripheral blood-derived mononuclear cells that attach to fibronectin-coated dishes, endocytose DiI-acLDL, and bind Ulex-lectin were used as EPCs. EPCs have been shown to proliferate, differentiate into mature ECs, form tubelike structures in vitro, and contribute to postnatal neovascularization in vivo (2, 7, 19, 20). Identification of EC precursors, however, has been a source of controversy (35, 36). EPCs were initially isolated from enriched CD34 antigen-positive (CD34⁺) cells from human peripheral blood (6). CD34 has been used as an EPC marker, and other markers such as AC133 and KDR were subsequently proposed as additional tools to further purify EPCs (16, 44). Recent studies, however, have demonstrated that not only CD34⁺ cells but also CD34^- cells can function as EPCs; bone marrow- or peripheral blood-derived CD34^- mononuclear cells readily differentiate into EC-like cells in culture and contribute to neovascularization in vivo (17, 38). It has also been shown that EPCs can develop from human peripheral blood CD14-positive monocytes (15, 37). To date, various criteria for EPC selection have been used, including DiI-acLDL(+)-Ulex-lectin(+), CD34⁺ (39), CD34⁺A133⁺KDR⁺ (34), CD34^-CD14⁺ (17), DiI-acLDL(+)vWF⁺ (29), and CD31⁺ (21). In this regard, the present data can be said to have been derived from this population of EPCs; peripheral blood derived DiI-acLDL(+)Ulex-lectin(+) adherent mononuclear cells.

The results of this study may have clinical relevance. Application of mechanical stress, such as shear stress or cyclic strain, during the production of artificial vascular grafts from vascular cells grown on a polymer matrix has recently been shown to enhance the development and function of the grafts (31). These engineered grafts exhibit superior mechanical strength and good patency when implanted in miniature swine. More recently, injection of muscles with EPCs isolated from human cord blood was shown to enhance neovascularization in the ischemic legs of nude mice (30). Thus manipulating EPCs by means of mechanical stress may be useful in the development of highly efficient tissue-engineered vessels or for the maturation of EPC cultures outside the body for cell therapy in ischemic vascular diseases.

	DISCLOSURES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was partly supported by Grants-in-Aid for Scientific Research, for Scientific Research on Priority Areas, and for Special Coordination Funds for Promoting Science and Technology from the Japanese Ministry of Education, Culture, Sports, Science and Technology; by a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare; and by Hayashi Memorial Foundation for Female Natural Scientists.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. Yamamoto, Dept. of Biomedical Engineering, Graduate School of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan (E-mail: k-yamamoto{at}umin.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Akimoto S, Mitsumata M, Sasaguri T, and Yoshida Y. Laminar shear stress inhibits vascular endothelial cell proliferation by inducing cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1). Circ Res 86: 185-190, 2000.[Abstract/Free Full Text]
2. Akita T, Murohara T, Ikeda H, Sasaki K, Shimada T, Egami K, and Imaizumi T. Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization. Lab Invest 83: 65-73, 2003.[Web of Science][Medline]
3. Ando J, Komatsuda T, Ishikawa C, and Kamiya A. Fluid shear stress enhanced DNA synthesis in cultured endothelial cells during repair of mechanical denudation. Biorheology 27: 675-684, 1990.[Web of Science][Medline]
4. Ando J, Nomura H, and Kamiya A. The effect of fluid shear stress on the migration and proliferation of cultured endothelial cells. Microvasc Res 33: 62-70, 1987.[Web of Science][Medline]
5. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, Kearne M, Magner M, and Isner JM. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 85: 221-228, 1999.[Abstract/Free Full Text]
6. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, and Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964-967, 1997.[Abstract/Free Full Text]
7. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, and Isner JM. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 18: 3964-3972, 1999.[Web of Science][Medline]
8. Bach TL, Barsigian C, Chalupowicz DG, Busler D, Yaen CH, Grant DS, and Martinez J. VE-cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels. Exp Cell Res 238: 324-334, 1998.[Web of Science][Medline]
9. Carmeliet P, Lampugnani MG, Moons L, Breviario F, Compernolle V, Bono F, Balconi G, Spagnuolo R, Oostuyse B, Dewerchin M, Zanetti A, Angellilo A, Mattot V, Nuyens D, Lutgens E, Clotman F, de Ruiter MC, Gittenberger-de Groot A, Poelmann R, Lupu F, Herbert JM, Collen D, and Dejana E. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98: 147-157, 1999.[Web of Science][Medline]
10. Carmeliet P and Luttun A. The emerging role of the bone marrow-derived stem cells in (therapeutic) angiogenesis. Thromb Haemost 86: 289-297, 2001.[Web of Science][Medline]
11. Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S, and Shyy JY. Mechanotransduction in response to shear stress. Roles of receptor tyrosine kinases, integrins, and Shc. J Biol Chem 274: 18393-18400, 1999.[Abstract/Free Full Text]
12. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 75: 519-560, 1995.[Abstract/Free Full Text]
13. Dewey CF. Effects of fluid flow on living vascular cells. J Biomech Eng 106: 31-35, 1984.[Web of Science][Medline]
14. Eskin SG, Ives CL, McIntire LV, and Navarro LT. Response of cultured endothelial cells to steady flow. Microvasc Res 28: 87-94, 1984.[Web of Science][Medline]
15. Fernandez Pujol B, Lucibello FC, Gehling UM, Lindemann K, Weidner N, Zuzarte ML, Adamkiewicz J, Elsasser HP, Muller R, and Havemann K. Endothelial-like cells derived from human CD14 positive monocytes. Differentiation 65: 287-300, 2000.[Web of Science][Medline]
16. Gehling UM, Ergun S, Schumacher U, Wagener C, Pantel K, Otte M, Schuch G, Schafhausen P, Mende T, Kilic N, Kluge K, Schafer B, Hossfeld DK, and Fiedler W. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood 95: 3106-3112, 2000.[Abstract/Free Full Text]
17. Harraz M, Jiao C, Hanlon HD, Hartley RS, and Schatteman GC. CD34^- blood-derived human endothelial cell progenitors. Stem Cells 19: 304-312, 2001.[Web of Science][Medline]
18. Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, Moore MA, and Rafii S. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J Exp Med 193: 1005-1014, 2001.[Abstract/Free Full Text]
19. Kalka C, Masuda H, Takahashi T, Gordon R, Tepper O, Gravereaux E, Pieczek A, Iwaguro H, Hayashi SI, Isner JM, and Asahara T. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res 86: 1198-1202, 2000.[Abstract/Free Full Text]
20. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, and Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 97: 3422-3427, 2000.[Abstract/Free Full Text]
21. Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C, Uchida S, Masuo O, Iwaguro H, Ma H, Hanley A, Silver M, Kearney M, Losordo DW, Isner JM, and Asahara T. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 107: 461-468, 2003.[Abstract/Free Full Text]
22. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, and Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7: 430-436, 2001.[Web of Science][Medline]
23. Kosaki K, Ando J, Korenaga R, Kurokawa T, and Kamiya A. Fluid shear stress increases the production of granulocyte-macrophage colony-stimulating factor by endothelial cells via mRNA stabilization. Circ Res 82: 794-802, 1998.[Abstract/Free Full Text]
24. Levesque MJ and Nerem RM. The elongation and orientation of cultured endothelial cells in response to shear stress. J Biomech Eng 107: 341-347, 1985.[Web of Science][Medline]
25. Levesque MJ, Sprague EA, Schwartz CJ, and Nerem RM. The influence of shear stress on cultured vascular endothelial cells: the stress response of an anchorage-dependent mammalian cell. Biotechnol Prog 5: 1-8, 1989.
26. Lin Y, Weisdorf DJ, Solovey A, and Hebbel RP. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 105: 71-77, 2000.[Web of Science][Medline]
27. Lyden D, Hattori K, Dias S, Costa C, Blaikie P, Butros L, Chadburn A, Heissig B, Marks W, Witte L, Wu Y, Hicklin D, Zhu Z, Hackett NR, Crystal RG, Moore MA, Hajjar KA, Manova K, Benezra R, and Rafii S. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 7: 1194-1201, 2001.[Web of Science][Medline]
28. Masuda H, Kawamura K, Tohda K, Shozawa T, Sageshima M, and Kamiya A. Increase in endothelial cell density before artery enlargement in flow-loaded canine carotid artery. Arteriosclerosis 9: 812-823, 1989.[Abstract/Free Full Text]
29. Moore MA, Hattori K, Heissig B, Shieh JH, Dias S, Crystal RG, and Rafii S. Mobilization of endothelial and hematopoietic stem and progenitor cells by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and angiopoietin-1. Ann NY Acad Sci 938: 36-47, 2001.[Web of Science][Medline]
30. Murohara T, Ikeda H, Duan J, Shintani S, Sasaki K, Eguchi H, Onitsuka I, Matsui K, and Imaizumi T. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 105: 1527-1536, 2000.[Web of Science][Medline]
31. Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, and Langer R. Functional arteries grown in vitro. Science 284: 489-493, 1999.[Abstract/Free Full Text]
32. Nomura H, Ishikawa C, Komatsuda T, Ando J, and Kamiya A. A disk-type apparatus for applying fluid shear stress on cultured endothelial cell. Biorheology 25: 461-470, 1988.[Web of Science][Medline]
33. Noria S, Cowan DB, Gotlieb AI, and Langille BL. Transient and steady-state effects of shear stress on endothelial cell adherens junctions. Circ Res 85: 504-514, 1999.[Abstract/Free Full Text]
34. Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA, and Rafii S. Expression of VEGFR-2 and AC133 by circulating human CD34⁺ cells identifies a population of functional endothelial precursors. Blood 95: 952-958, 2000.[Abstract/Free Full Text]
35. Rabbany SY, Heissig B, Hattori K, and Rafii S. Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization. Trends Mol Med 9: 109-117, 2003.[Web of Science][Medline]
36. Rafii S. Circulating endothelial precursors: mystery, reality, and promise. J Clin Invest 105: 17-19, 2000.[Web of Science][Medline]
37. Rehman J, Li J, Orschell CM, and March KL. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 107: 1164-1169, 2003.[Abstract/Free Full Text]
38. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, and Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109: 337-346, 2002.[Web of Science][Medline]
39. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, and Hammond WP. Evidence for circulating bone marrow-derived endothelial cells. Blood 92: 362-367, 1998.[Abstract/Free Full Text]
40. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, and Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 5: 434-438, 1999.[Web of Science][Medline]
41. Vittet D, Buchou T, Schweitzer A, Dejana E, and Huber P. Targeted null-mutation in the vascular endothelial-cadherin gene impairs the organization of vascular-like structures in embryoid bodies. Proc Natl Acad Sci USA 94: 6273-6278, 1997.[Abstract/Free Full Text]
42. Wang DM and Tarbell JM. Modeling interstitial flow in an artery wall allows estimation of wall shear stress on smooth muscle cells. J Biomech Eng 117: 358-363, 1995.[Web of Science][Medline]
43. Yang S, Graham J, Kahn JW, Schwartz EA, and Gerritsen ME. Functional roles for PECAM-1 (CD31) and VE-cadherin (CD144) in tube assembly and lumen formation in three-dimensional collagen gels. Am J Pathol 155: 887-895, 1999.[Abstract/Free Full Text]
44. Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J, and Buck DW. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90: 5002-5012, 1997.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Obi, K. Yamamoto, N. Shimizu, S. Kumagaya, T. Masumura, T. Sokabe, T. Asahara, and J. Ando Fluid shear stress induces arterial differentiation of endothelial progenitor cells J Appl Physiol, January 1, 2009; 106(1): 203 - 211. [Abstract] [Full Text] [PDF]

				T. N. Milovanova, V. M. Bhopale, E. M. Sorokina, J. S. Moore, T. K. Hunt, M. Hauer-Jensen, O. C. Velazquez, and S. R. Thom Lactate Stimulates Vasculogenic Stem Cells via the Thioredoxin System and Engages an Autocrine Activation Loop Involving Hypoxia-Inducible Factor 1 Mol. Cell. Biol., October 15, 2008; 28(20): 6248 - 6261. [Abstract] [Full Text] [PDF]

				A. Zampetaki, J. P. Kirton, and Q. Xu Vascular repair by endothelial progenitor cells Cardiovasc Res, June 1, 2008; 78(3): 413 - 421. [Abstract] [Full Text] [PDF]

				Q. Xu Stem Cells and Transplant Arteriosclerosis Circ. Res., May 9, 2008; 102(9): 1011 - 1024. [Abstract] [Full Text] [PDF]

				K. Kawasaki, T. Watabe, H. Sase, M. Hirashima, H. Koide, Y. Morishita, K. Yuki, T. Sasaoka, T. Suda, M. Katsuki, et al. Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells J. Cell Biol., April 3, 2008; 181(1): 131 - 141. [Abstract] [Full Text] [PDF]

				N. Shimizu, K. Yamamoto, S. Obi, S. Kumagaya, T. Masumura, Y. Shimano, K. Naruse, J. K. Yamashita, T. Igarashi, and J. Ando Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle cells by activating PDGF receptor {beta} J Appl Physiol, March 1, 2008; 104(3): 766 - 772. [Abstract] [Full Text] [PDF]

				A. M. D. Malone, C. T. Anderson, P. Tummala, R. Y. Kwon, T. R. Johnston, T. Stearns, and C. R. Jacobs Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism PNAS, August 14, 2007; 104(33): 13325 - 13330. [Abstract] [Full Text] [PDF]

				L. Zeng, Q. Xiao, A. Margariti, Z. Zhang, A. Zampetaki, S. Patel, M. C. Capogrossi, Y. Hu, and Q. Xu HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells J. Cell Biol., September 25, 2006; 174(7): 1059 - 1069. [Abstract] [Full Text] [PDF]

				H.C. de Boer, C. Verseyden, L.H. Ulfman, J.J. Zwaginga, I. Bot, E.A. Biessen, T.J. Rabelink, and A.J. van Zonneveld Fibrin and Activated Platelets Cooperatively Guide Stem Cells to a Vascular Injury and Promote Differentiation Towards an Endothelial Cell Phenotype Arterioscler Thromb Vasc Biol, July 1, 2006; 26(7): 1653 - 1659. [Abstract] [Full Text] [PDF]

				H. Wang, G. M. Riha, S. Yan, M. Li, H. Chai, H. Yang, Q. Yao, and C. Chen Shear Stress Induces Endothelial Differentiation From a Murine Embryonic Mesenchymal Progenitor Cell Line Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1817 - 1823. [Abstract] [Full Text] [PDF]

				J. I. Rotmans, J. M.M. Heyligers, H. J.M. Verhagen, E. Velema, M. M. Nagtegaal, D. P.V. de Kleijn, F. G. de Groot, E. S.G. Stroes, and G. Pasterkamp In Vivo Cell Seeding With Anti-CD34 Antibodies Successfully Accelerates Endothelialization but Stimulates Intimal Hyperplasia in Porcine Arteriovenous Expanded Polytetrafluoroethylene Grafts Circulation, July 5, 2005; 112(1): 12 - 18. [Abstract] [Full Text] [PDF]

				L. Rossig, C. Urbich, T. Bruhl, E. Dernbach, C. Heeschen, E. Chavakis, K.-i. Sasaki, D. Aicher, F. Diehl, F. Seeger, et al. Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells J. Exp. Med., June 6, 2005; 201(11): 1825 - 1835. [Abstract] [Full Text] [PDF]

				K. Yamamoto, T. Sokabe, T. Watabe, K. Miyazono, J. K. Yamashita, S. Obi, N. Ohura, A. Matsushita, A. Kamiya, and J. Ando Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1915 - H1924. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Free All Versions of this Article: 95/5/2081    most recent 00232.2003v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (49) Citing Articles via Google Scholar Google Scholar Articles by Yamamoto, K. Articles by Ando, J. Search for Related Content PubMed PubMed Citation Articles by Yamamoto, K. Articles by Ando, J.