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

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 19/9/1178 04-3261fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal 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 Google Scholar Google Scholar Articles by Sakao, S. Articles by Voelkel, N. F. Search for Related Content PubMed PubMed Citation Articles by Sakao, S. Articles by Voelkel, N. F.

Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells

Seiichiro Sakao^*,1, Laimute Taraseviciene-Stewart^*, Jong Deog Lee^*, Kathy Wood, Carlyne D. Cool and Norbert F. Voelkel^*

^* The Pulmonary Hypertension Center and
Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado, USA

¹ Correspondence: Division of Pulmonary Sciences and Critical Care Medicine, 4200 East Ninth Ave., C272, Denver, CO 80262, USA. E-mail: seiichirosakao{at}yahoo.co.jp

SPECIFIC AIMS

Because the pulmonary angioproliferation and development of severe pulmonary hypertension were preventable by treatment of rats with a caspase inhibitor in a new model of severe PH, we developed the hypothesis that endothelial cell growth and emergence of phenotypically altered vascular cells in severe PH are the consequence of initial apoptosis and subsequent selection of apoptosis-resistant proliferative vascular cells. VEGF is an obligatory endothelial cell growth factor, and we hypothesize that the combination of initial apoptosis induced by VEGF receptor blockade followed by high shear stress can generate apoptosis-resistant proliferative endothelial cells.

PRINCIPAL FINDINGS

1. Endothelial cell proliferation in the CELLMAX system
We adapted CELLMAX artificial capillary modules to analyze the effects of the VEGF receptor blocker SU5416 on human pulmonary microvascular endothelial cells (HPMVEC) under pulsatile shear stress at high (10.1 dynes/cm²) or low (1.9 dynes/cm²) shear stress. Cells expressing processed or active caspase-3 are likely undergoing apoptosis. Active caspase-3 immunostaining was detected in HPMVEC cultured in CELLMAX artificial capillary modules 7 days after SU5416 addition whether SU5416 had been added or not. On day 7 there was a decrease in the number of active caspase-3-positive cells of SU5416-treated cells compared with control cells. Cells expressing PCNA are likely actively proliferating. PCNA immunostaining was detected in HPMVEC cultured in SU5416-treated and -untreated cells. There was an increase on day 7 in the number of PCNA-positive cells of the SU5416-treated cells compared with control cells, suggesting that SU-5416, not shear stress, induces the hyperproliferative state of these endothelial cells.

2. Detection of initial apoptosis induced by SU5416
To determine whether initial apoptosis is induced by the VEGF receptor blocker SU5416, we investigated the time course of cell growth under high shear stress. Active caspase-3-positive staining was detected in HPMVEC cultured 1 day after SU5416 addition and 3 days after SU5416 addition; there was a decrease in the number of active caspase-3-positive staining cells and an increase in the number of PCNA-positive staining cells on day 7 after SU5416 addition.

3. Quantification of apoptotic and proliferating endothelial cells
The number of apoptotic and proliferating cells was assessed by flow cytometry. Apoptosis was assessed by annexin V staining. Annexin V is a 35-36 kDa Ca²⁺-dependent phospholipid-binding protein with a high affinity for phosphatidylserine (PS). Annexin V labeled with a fluorophore can identify apoptotic cells by binding to PS exposed on the outer cell membrane leaflet. There was an increase in the number of annexin V-positive cells treated with SU5416 under high shear stress on day 1 but not in cells treated with SU5416 alone or exposed only to shear (Fig. 1 A). Bromodeoxyuridine (BrdU), an analog of the DNA precursor thymidine, is incorporated into newly synthesized DNA by cells entering and progressing through the S (DNA synthesis) phase of the cell cycle. We found an increase in the number of BrdU-positive cells treated with SU5416 under high shear stress on day 7 but no increase in cells treated with SU5416 but not exposed to shear stress or in cells exposed to shear alone (Fig. 1B ).

View larger version (20K): [in this window] [in a new window]   Figure 1. A) Quantification of apoptotic endothelial cells and inhibition of SU5416-induced apoptosis by the caspase family inhibitor IV. The number of apoptotic cells was measured by flow cytometry. Cells undergoing apoptosis were assessed by annexin V staining. There was an increase in the number of annexin V-positive sheared cells at day 1 after SU5416 addition () but no increase after SU5416 addition alone () or shear stress without SU5416 addition (). Data points represent the means of 3 independent experiments. *P < 0.05 vs. SU5416 or high shear stress, n 3. B) Quantification of proliferating endothelial cells and blockade of proliferation by the caspase family inhibitor IV. Individual cells synthesizing DNA were determined by the immunofluorescent staining of incorporated BrdU and flow cytometric analysis. Incorporated BrdU was stained with specific anti-BrdU fluorescent antibodies. Levels of cell-associated BrdU were measured by flow cytometry. There was an increase in the number of BrdU-positive cells at day 7 after SU5416 addition (), but no increase in cells where no SU5416 was added () and no increase in sheared, SU5416-added, and caspase family inhibitor blocked cells (•). Data points represent the means of 3 independent experiments. *P < 0.05 vs. SU5416 or high shear stress, n 3.

4. Inhibition of SU5416-induced apoptosis and blockade of cell proliferation by a caspase inhibitor (Z-Asp-2, 6 dichlorobenzoyloxymethylketone)
This inhibitor of caspases inhibits IL-1ß-converting enzyme (ICE) and apoptotic cell death. The caspase inhibitor prevented the increase in the number of annexin V-positive cells on day 1 (Fig. 1A ) as well as the increase of BrdU-positive cells on day 7 after SU5416 treatment (Fig. 1B ).

5. Selection of apoptosis-resistant, proliferating endothelial cells by VEGF receptor blockade
HPMVEC exposed to the combination of high shear, TNF- + cycloheximide for 8 h or to H₂O₂ for 16 h (Fig. 2 Ab ) on day 7 underwent apoptosis as assessed by the annexin V propidium iodide binding assay. However, the combination of TNF- + cycloheximide or H₂O₂ did cause apoptosis to a lesser degree when the cells had been treated on day 1 with SU5416. Addition of the caspase inhibitor on day 1 did not block the TNF- + cycloheximide-induced apoptosis or the H₂O₂-induced apoptosis rate on day 7. In contrast, day 1 SU5416-treated cells demonstrated a low apoptosis rate when challenged with TNF- + cycloheximide or H₂O₂. (see online supplement.)

View larger version (29K): [in this window] [in a new window]   Figure 2. Selection of an apoptosis-resistant, proliferating endothelial cell phenotype by SU5416. Selection of an apoptosis-resistant cell phenotype not dependent on serum growth factors. HPMVECs cultured in serum-free medium underwent apoptosis as assessed by annexin V propidium iodide binding assay. Data represent the mean of 3 independent experiments; P < 0.05.

6. Apoptosis-resistant cells documented by exposure to serum-free medium
HPMVECs cultured in serum-free medium underwent apoptosis as assessed by the annexin V propidium iodide binding assay (Fig. 2 ). We compared cells exposed to low or high shear stress and induced apoptosis by changing the CELLMAX perfusate to a serum-free medium. Compared with cells exposed to low shear stress or cells exposed to high shear stress without SU5416 treatment on day 1, cells exposed to high shear stress and SU5416 on day 1 demonstrated a low apoptosis rate, indicating that the initial apoptosis induced by SU5416 resulted in selection of a serum-independent cell phenotype.

CONCLUSIONS AND SIGNIFICANCE

Our experiments with HPMVEC seeded in the artificial capillary system demonstrated that a combined VEGF I and II receptor blocker (SU5416) induces apoptosis. When this VEGF receptor blockade-induced apoptosis was followed by high fluid shear stress, a hyperproliferative state was generated; within 7 days phenotypically altered endothelial cells emerged. These altered endothelial cells expressed the tumor marker survivin and the antiapoptotic protein Bcl-_XL, and were resistant to induction of apoptosis after challenge with TNF- + cycloheximide or hydrogen peroxide; the cells also demonstrated survival in serum-free culture medium. This apoptosis-resistant endothelial cell phenotype emerged without any drug use after addition of apoptosed endothelial cells to the artificial capillary system. Our data support the concept that apoptosis induces apoptosis-resistant, proliferative endothelial cells.

Endothelial cells seeded into the CELLMAX system grow and coat the lumina of the artificial capillaries. Using immunohistochemistry, we found relatively few caspase-3-positive cells with SU5416 addition under low or high shear stress conditions on day 7. Using annexin V as a marker, the peak of apoptosis was found on day 1 after SU5416 addition, when shear stress was high (Fig. 1A ). There was a trend toward an increase of BrdU-positive endothelial cells between day 1 and day 7 of the high-flow experiments (Fig. 1B ), yet the number of BrdU-positive cells increased on day 7 of the experiment compared with day 1 or 5 (Fig. 1B ). This indicates that shear stress by itself is insufficient to cause exuberant endothelial cell proliferation in the CELLMAX system and that the apoptosis induced by SU5416 is important for the subsequent cell proliferation. This notion is supported by data that show inhibition of both the initial SU5416-induced apoptosis and subsequent cell proliferation by the caspase inhibitor.

To test whether the SU5416-treated hyperproliferative cells were apoptosis resistant, we added TNF- + cycloheximide or hydrogen peroxide to the perfusate of the artificial capillary system. Cells treated on day 1 with SU5416, which induced initial apoptosis, developed significantly less subsequent apoptosis than those not exposed to the VEGF receptor blocker. An additional nonpharmacological approach was to switch the cells to serum- and growth factor-free medium; again, SU5416-treated cells were relatively apoptosis resistant (Fig. 2) .

Our data reflect the paradox that growth factor inhibition fosters the emergence of apoptosis-resistant and hyperproliferative cells. This paradox has recently been described in experiments that concluded that there is "life after corpse engulfment." It was shown that cells with apoptosis induced by UV irradiation (after they had been phagocytosed by other cells) released growth factors into the culture medium and that this conditioned medium made naive epithelial or endothelial cells apoptosis resistant.

In our shear stress experiments, whether the SU5416-treated apoptotic cells were phagocytosed by neighboring cells of the CELLMAX system was not examined. Most cell types (not only professional phagocytes such as macrophages) have the ability to phagocytose apoptosed cells, and we consider this a possibility. It is unclear why the VEGF receptor blockade does not induce apoptosis in all of the endothelial cells or whether the surviving cells do so because they respond to survival signals that may be released by the dying cells (Fig. 2B ). It is conceivable that the endothelial cells contain some apoptosis-resistant precursor cells that expand under the conditions of our experiments.

We continued to characterize phenotypical alterations using Western blot analysis. These data show that high shear stress causes increased expression of phospho-Akt-1 and of the antiapoptotic Bcl-_XL protein. High shear stress also caused expression of survivin and reduced the expression of caveolin-1. Survivin and caveolin-1 were included in this survey because of our recent finding of decreased or lost expression of caveolin-1 and -2 in plexiform lesions of patients with severe PH and expression of survivin in these lesions. Loss of caveolin-1 expression has been associated with cancers, and survivin expression has been demonstrated in lung, colon, prostate, and breast cancers. A recent report noted a reduction of caveolin-1 in pulmonary endothelial cells in the monocrotaline rat model of pulmonary hypertension. We believe our data are the first to show that high shear stress can express the tumor marker survivin in proliferative endothelial cells.

Of interest, the VEGF receptor blocker SU5416 does not cause prolonged apoptosis of endothelial cells (Fig. 1) , indicating that the cells become resistant to the drug and their survival VEGF independent.

Our experimental model supports the concept that apoptosis-resistant hyperproliferative endothelial cells can emerge at shear stress-sensitive sites in the lung circulation in severe pulmonary hypertension. Although we do not address experimentally the factor(s) that confer apoptosis resistance and phenotypical alterations of a subpopulation of endothelial cells, we suggest that blockade of the signal transduction of the obligatory survival factor VEGF, in combination with high shear, provides a selection pressure. It is possible that the surviving and proliferating cells are precursor cells.

View larger version (37K): [in this window] [in a new window]   Figure 3. Sequence of events that lead from initial apoptosis to proliferation of apoptosis-resistant endothelial cells. The combination of initial apoptosis induced by VEGF receptor blockade and high fluid shear stress generates apoptosis-resistant proliferative endothelial cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3261fje; doi: 10.1096/fj.04-3261fje

This article has been cited by other articles:

				E. D. Michelakis, M. R. Wilkins, and M. Rabinovitch Emerging Concepts and Translational Priorities in Pulmonary Arterial Hypertension Circulation, September 30, 2008; 118(14): 1486 - 1495. [Full Text] [PDF]

				P. R. Rai, C. D. Cool, J. A. C. King, T. Stevens, N. Burns, R. A. Winn, M. Kasper, and N. F. Voelkel The Cancer Paradigm of Severe Pulmonary Arterial Hypertension Am. J. Respir. Crit. Care Med., September 15, 2008; 178(6): 558 - 564. [Abstract] [Full Text] [PDF]

				N. Amabile, C. Heiss, W. M. Real, P. Minasi, D. McGlothlin, E. J. Rame, W. Grossman, T. De Marco, and Y. Yeghiazarians Circulating Endothelial Microparticle Levels Predict Hemodynamic Severity of Pulmonary Hypertension Am. J. Respir. Crit. Care Med., June 1, 2008; 177(11): 1268 - 1275. [Abstract] [Full Text] [PDF]

				L. Moreno-Vinasco, M. Gomberg-Maitland, M. L. Maitland, A. A. Desai, P. A. Singleton, S. Sammani, L. Sam, Y. Liu, A. N. Husain, R. M. Lang, et al. Genomic assessment of a multikinase inhibitor, sorafenib, in a rodent model of pulmonary hypertension Physiol Genomics, April 21, 2008; 33(2): 278 - 291. [Abstract] [Full Text] [PDF]

				S. Mukhopadhyay, M. Shah, F. Xu, K. Patel, R. M. Tuder, and P. B. Sehgal Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L449 - L468. [Abstract] [Full Text] [PDF]

				S. L. Archer, M. Gomberg-Maitland, M. L. Maitland, S. Rich, J. G. N. Garcia, and E. K. Weir Mitochondrial metabolism, redox signaling, and fusion: a mitochondria-ROS-HIF-1{alpha}-Kv1.5 O2-sensing pathway at the intersection of pulmonary hypertension and cancer Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H570 - H578. [Abstract] [Full Text] [PDF]

				S. Sakao, L. Taraseviciene-Stewart, C. D. Cool, Y. Tada, Y. Kasahara, K. Kurosu, N. Tanabe, Y. Takiguchi, K. Tatsumi, T. Kuriyama, et al. VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells FASEB J, November 1, 2007; 21(13): 3640 - 3652. [Abstract] [Full Text] [PDF]

				F. A. Masri, W. Xu, S. A. A. Comhair, K. Asosingh, M. Koo, A. Vasanji, J. Drazba, B. Anand-Apte, and S. C. Erzurum Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L548 - L554. [Abstract] [Full Text] [PDF]

				L. Taraseviciene-Stewart, M. R. Nicolls, D. Kraskauskas, R. Scerbavicius, N. Burns, C. Cool, K. Wood, J. E. Parr, S. A. Boackle, and N. F. Voelkel Absence of T Cells Confers Increased Pulmonary Arterial Hypertension and Vascular Remodeling Am. J. Respir. Crit. Care Med., June 15, 2007; 175(12): 1280 - 1289. [Abstract] [Full Text] [PDF]

				F. Zwerts, F. Lupu, A. De Vriese, S. Pollefeyt, L. Moons, R. A. Altura, Y. Jiang, P. H. Maxwell, P. Hill, H. Oh, et al. Lack of endothelial cell survivin causes embryonic defects in angiogenesis, cardiogenesis, and neural tube closure Blood, June 1, 2007; 109(11): 4742 - 4752. [Abstract] [Full Text] [PDF]

				S. Zhang, H. H. Patel, F. Murray, C. V. Remillard, C. Schach, P. A. Thistlethwaite, Paul. A. Insel, and J. X.-J. Yuan Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1202 - L1210. [Abstract] [Full Text] [PDF]

				M. Levy, C. Maurey, D. S. Celermajer, P. R. Vouhe, C. Danel, D. Bonnet, and D. Israel-Biet Impaired Apoptosis of Pulmonary Endothelial Cells Is Associated With Intimal Proliferation and Irreversibility of Pulmonary Hypertension in Congenital Heart Disease J. Am. Coll. Cardiol., February 20, 2007; 49(7): 803 - 810. [Abstract] [Full Text] [PDF]

				S. Sakao, L. Taraseviciene-Stewart, K. Wood, C. D. Cool, and N. F. Voelkel Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L362 - L368. [Abstract] [Full Text] [PDF]

				D. C. Altieri Targeted therapy by disabling crossroad signaling networks: the survivin paradigm. Mol. Cancer Ther., March 1, 2006; 5(3): 478 - 482. [Abstract] [Full Text] [PDF]

				E. D. Michelakis Spatio-Temporal Diversity of Apoptosis Within the Vascular Wall in Pulmonary Arterial Hypertension: Heterogeneous BMP Signaling May Have Therapeutic Implications Circ. Res., February 3, 2006; 98(2): 172 - 175. [Full Text] [PDF]

				N. F. Voelkel, R. W. Vandivier, and R. M. Tuder Vascular endothelial growth factor in the lung Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L209 - L221. [Abstract] [Full Text] [PDF]

				C. D. Cool, S. D. Groshong, J. Oakey, and N. F. Voelkel Pulmonary Hypertension: Cellular and Molecular Mechanisms Chest, December 1, 2005; 128(6_suppl): 565S - 571S. [Full Text] [PDF]

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 19/9/1178 04-3261fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal 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 Google Scholar Google Scholar Articles by Sakao, S. Articles by Voelkel, N. F. Search for Related Content PubMed PubMed Citation Articles by Sakao, S. Articles by Voelkel, N. F.