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Published as doi: 10.1096/fj.07-8432com.
 (The FASEB Journal. 2007;21:3640-3652.)
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VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells

Seiichiro Sakao*,{ddagger},1, Laimute Taraseviciene-Stewart*, Carlyne D. Cool{dagger}, Yuji Tada{ddagger}, Yasunori Kasahara{ddagger}, Katsushi Kurosu{ddagger}, Nobuhiro Tanabe{ddagger}, Yuichi Takiguchi{ddagger}, Koichiro Tatsumi{ddagger}, Takayuki Kuriyama{ddagger} and Norbert F. Voelkel*

* Pulmonary Hypertension Center and

{dagger} Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado, USA; and

{ddagger} Department of Respirology (B2), Graduate School of Medicine, Chiba University, Chiba, Japan

1Correspondence: Department of Respirology (B2), Graduate School of Medicine, Chiba University, 1–8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: sakaos{at}faculty.chiba-u.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Severe pulmonary hypertension (PH) is characterized by complex precapillary arteriolar lesions, which contain phenotypically altered smooth muscle (SM) and endothelial cells (EC). We have demonstrated that VEGF receptor blockade by SU5416 {3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-indolin 2-one} in combination with chronic hypoxia causes severe angioproliferative PH associated with arterial occlusion in rats. We postulate that endothelial-mesenchymal transdifferentiation can take place in the occlusive lesions and that endothelium-derived mesenchymal cells can further differentiate toward a SM phenotype. To examine this hypothesis, we incubated human pulmonary microvascular endothelial cells (HPMVEC) with SU5416 and analyzed these cells utilizing quantitative-PCR, immunofluorescent staining and flow cytometry analysis. In vitro studies in HPMVEC demonstrated that SU5416 suppressed PGI2S gene expression while potently inducing COX-2, VEGF, and TGF-ß1 expression; and caused transdifferentiation of mature vascular endothelial cells (defined by Dil-ac-LDL, Lectin and Factor VIII) to SM-like (as defined by expression of {alpha}-SM actin) "transitional" cells, coexpressing both endothelial and SM markers. SU5416 expanded the number of CD34 and/or c-kit positive cells and caused transdifferentiation of CD34 positive cells but not negative cells. In conclusion, our data show that SU5416 generated a selection pressure that killed some EC and expanded progenitor-like cells to transdifferentiate to SM-like and neuronal-like cells.—Sakao, S., Taraseviciene-Stewart, L., Cool, C. D., Tada, Y., Kasahara, Y., Kurosu, K., Tanabe, N., Takiguchi, Y., Tatsumi, K., Kuriyama, T., and Voelkel, N. F. VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells.


Key Words: pulmonary hypertension • human pulmonary microvascular endothelial cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) is an obligatory survival factor for endothelial cells (EC) since VEGF withdrawal from cultured EC induces apoptosis (1) . VEGF signaling in EC occurs principally through two tyrosine kinase receptors VEGF-R I (Flt) and VEGF-R II (Flk/KDR), and knockout of the genes encoding VEGF, VEGF-R I, and VEGF-R II are embryonic lethal (2) . Taken together, these findings are consistent with the concept that VEGF is essential for normal angiogenesis to proceed. However, overexpression of VEGF causes EC proliferation, formation of glomeruloid structures in the skin (3) or hypervascularization in the mouse trachea (4) . Thus withdrawal of VEGF or impaired signaling causes lung vessel loss as shown by Kasahara et al. (5) or recently by Tang et al. (6) and also capillary regression in skeletal muscle (7) . However, in contrast, overexpression of VEGF is associated with tumor vessel growth (8) and EC proliferation in severe pulmonary hypertension (9) .

Severe angioproliferative pulmonary hypertension (PH) is characterized by complex pulmonary precapillary arteriolar lesions (10 11 12 13) , which contain phenotypically altered smooth muscle cells (SMC) and EC (11) . In addition to lumen-obliterating cell aggregates, which form the so-called plexiform lesions, muscularized arteries are also frequently present. Vasoconstriction as well as peptide (endothelin and angiotensin II) and nonpeptide (serotonin) growth factors have been postulated to be responsible for the muscularization of the pulmonary arteries in severe PH (14 15 16) . Here we hypothesize that an additional or alternative mechanism contributing to the vascular media hypertrophy may be transdifferentiation of pulmonary endothelial cells. Indeed "transitional cells" demonstrating features of both EC and vascular smooth muscle cells (VSMC) have been identified in the plexiform lesions in the lungs from patients with severe angioproliferative PH (18) . Transforming growth factor-beta 1 (TGF-ß1) has been shown to induce the transdifferentiation of cultured aortic EC into a smooth muscle (SM)-like phenotype (19) . Mature bovine systemic and pulmonary EC contain cell populations that can acquire a SM phenotype via a transdifferentiation process that is TGF-ß1- and cell-cell contact-dependent, but proliferation-independent (17) . Recently Sca-1+ progenitor cells that reside in the vascular adventitia have also been shown to transdifferentiate into SM-like neointimal cells (20) . Transdifferentiation of normal lung microvascular cells has not been previously reported, nor is it known whether the adult lung contains resident endothelial precursor cells that could potentially participate in the repair of the injured lung.

We have demonstrated that VEGF receptor blockade with the VEGF-RI/VEGF-R II antagonist SU5416 combined with chronic hypoxic vasoconstriction results in angioproliferative pulmonary hypertension in adult rats (21) , and in vitro in EC, under conditions of increased fluid shear stress, causes initial apoptosis followed by exuberant proliferation of the surviving EC (22) . Here we ask the question: What is the nature of the surviving cells? Because VEGF is an obligatory survival factor for EC (1) , we made use of the effective VEGF-R antagonist to induce pulmonary microvascular EC apoptosis in order to generate a selection pressure for the EC and to track the fate of the surviving EC.

We report here that the EC that survive the VEGF-R blockade-induced apoptosis are multipotent, likely bone marrow-derived precursor cells (23) that transdifferentiate into vascular smooth muscle cells (VSMC)-like cells. This is the first report that demonstrates the presence of resident multipotent vascular precursor cells in adult lungs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and reagents
Human pulmonary microvascular endothelial cells (HPMVEC) were from Clonetics (Baltimore, MD, USA). They were cultured in endothelial cell growth medium (EGM) supplemented with 5% fetal bovine serum (Clonetics). The VEGF-R antagonist SU5416 {3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-indolin 2-one} was provided by SUGEN Inc (South San Francisco, CA, USA). SU5416 was suspended in CMC [0.5% (w/v) carboxymethylcellulose sodium, 0.9% (w/v) sodium chloride, 0.4% (v/v) polysorbate 80, and 0.9% (v/v) benzyl alcohol in deionized water]. The cells at passage 7 were incubated with SU5416 (10 µM) or with vehicle (CMC), plus-minus TGF-ß1 neutralizing antibodies (Ab) and VEGF neutralizing Ab and cultured until passage 12. At each passage the cells were seeded at a density of 1.5 x 104 cells/cm2 and were subcultured to 70–90% confluency (4–8 days). DiI-Ac-LDL (1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine-labeled Ac-LDL) was from Biomedical Technologies (Stoughton, MA, USA, USA). FITC-labeled Ulex europaeus agglutinin-I (lectin) was from Sigma (St. Louis, MO, USA). The following Ab were used: mouse anti-{alpha}-SM-actin Ab (1:1000, Sigma), mouse antivimentin Ab (1:200, DAKO, Carpinteria, CA, USA), mouse anti-human desmin Ab (1:100, DAKO), anti-mouse IgG Ab conjugated with Alexa-594 fluorescent dye (1:500, Molecular Probes, Eugene, OR, USA), rabbit antivon Willebrand factor Ab (1:1000, DAKO), anti-rabbit IgG Ab conjugated with Alexa-488 fluorescent dye (1:500, Molecular Probes, Eugene, OR, USA), anti-rabbit Tyrosine Hydroxylase (TH) Ab (BIOMOL International L.P., Plymouth Meeting, PA, USA) neutralizing Ab against active TGF-ß1 (AB-100-NA, R&D Systems, Minneapolis, MN, USA), neutralizing Ab against active VEGF (Sigma-Aldrich Corp., St. Louis, MO, USA), FITC-conjugated anti-CD34 Ab (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PE-conjugated anti-CD117 (c-kit) Ab (1:50, BD Biosciences Pharmingen, San Diego, CA, USA).

Quantitative RT-PCR
Quantitative RT-PCR was performed on a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using 2 x SYBR Green PCR Master Mix (Applied Biosystems). Primers were designed to meet specific criteria by using Primer Express V.1.0 software (Applied Biosystems). Total RNA was extracted from the cells treated with or without SU5416 using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. RNA (1 µg) was reverse-transcribed using random primer and MultiScribe RT (High-Capacity cDNA Archive Kit, Applied Biosystems). Assay-on-demand gene expression probes for PGI2S, COX-2, VEGF, TGF-ß1, and ß-actin were purchased from Applied Biosystems. PCR reactions were performed in 20 µl volumes containing 9 µl of cDNA, 10 µl of TaqMan Master Mix (Applied Biosystems), and 1 µl of assay-on-demand primer and probe. PCR amplifications (final volume 25 µl) were run in duplicate, using the following conditions: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Primers were used at a concentration of 200 nM in each reaction. Relative quantitation of gene expression was determined using the GeneAmp 5700 SDS software (Applied Biosystems).

Uptake of DiI-Ac-LDL and staining of Ulex europaeus agglutinin-I (lectin)
For uptake of DiI-Ac-LDL (Biomedical Technologies, Stoughton, MA, USA) cells were incubated with 4.0 µg/ml DiI-Ac-LDL for 4 h. Then the cells were fixed with 2% paraformaldehyde (PFA). For staining of Ulex europaeus agglutinin-I (lectin) cells were fixed with 2% PFA and incubated with 10 µg/ml FITC-labeled Ulex europaeus agglutinin-I (lectin; Sigma, St. Louis, MO, USA) for 1 h. These cells were examined with a ZEISS Axioskop 2 microscope with the KS300 imaging system (Carl Zeiss, Inc, Thornwood, NY, USA). Positive cells were counted in 3 different fields at a magnification of 200x in a fluorescence microscope.

Immunofluorescent staining
Cells were fixed in a 1:1 mixture of methanol and acetone for 2 min followed by blocking with normal goat serum for 30 min, and incubated with primary antibodies (anti-{alpha}-SM-actin, antivon Willebrand factor, antivimentin, and antidesmin) for 1 h at room temperature and with secondary antibodies (anti-mouse IgG Ab conjugated with Alexa-594 fluorescent dye and anti-rabbit IgG Ab conjugated with Alexa-488 fluorescent dye) for 1 h at room temperature. Incubation with antityrosine hydroxylase (TH) antibody was performed at 4°C for 16 h; secondary antibody was Alexa-488-conjugated anti-rabbit IgG. Stained cells were embedded in VectaShield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and examined with a ZEISS Axioskop 2 microscope with the KS300 imaging system. Positive cells were counted in 3 different fields at 200x magnification using a fluorescence microscope.

Flow cytometry
Cells were detached with trypsin (0.25 mg/ml) for 3 min at 37°C and incubated with FITC-conjugated CD34 (Santa Cruz Biotechnology) and/or PE-conjugated anti-CD117 (c-kit) Abs (1:50, BD Biosciences Pharmingen) antibodies. Analysis was performed on at least 5000 cells/sample in a FACScan using CellQuest software (BD Biosciences Pharmingen) and manual gating.

Magnetic cell sorting (MACS)
After trypsinization of HPMVEC at passage 6, 100 µl of FcR Blocking Reagent (Direct CD34 progenitor cell isolation kit, Miltenyi Biotec Inc, Auburn, CA, USA) per 108 total cells was added to the cell suspension to inhibit non-specific or Fc-receptor mediated binding of CD34 MicroBeads (Direct CD34 progenitor cell isolation kit, Miltenyi Biotec) to nontarget cells. Cells were labeled by adding 100 µl CD34 MicroBeads per 108 total cells and incubated for 30 min at 6–12°C. After washing, cells were resuspended in 500 µl buffer and applied to the MS+/RS+ column with the column adapter in the magnetic field of the MACS separator. The column was washed 3x with 500 µl buffer. The column was removed from the separator, and the retained cells were flushed out with 1 ml buffer under pressure using the plunger supplied with the column. The cells were incubated with or without SU5416 and cultured until passage 12.

Gene microarray analysis
Total RNA was extracted from the cells treated with or without SU5416 for 5 days using the RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions. Total RNA (15 µg) was collected from each sample and evaluated individually. Of the labeled cRNA mixture, 20 µg was applied to the GeneChip microarray analysis (Affymetrix, Santa Clara, CA, USA) as described previously (13 , 24 , 25) , and hybridization was performed for 18–20 h. Detailed protocols for data analysis of Affymetrix microarrays and extensive documentation of the sensitivity and quantitative aspects of the method have been described (26 27 28) .

Statistical analysis
Three independent experiments were subjected to statistical analysis. Results were expressed as mean ± SEM. Data were analyzed using Student’s t-test, as appropriate. A P < 0.05 was considered significant for all tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SU5416-induced transdifferentiation of HPMVEC to SM-like cells
All of the experiments were conducted with human pulmonary microvascular endothelial cells (HPMVEC). Cells at passage 7 were incubated with or without SU5416 for 5 days and cultured for 3 or 5 more passages.

We first established that the HPMVEC were free of contamination with VSMC by staining with Ulex europaeus agglutinin-I, examining the uptake of DiI-Ac-LDL and by immunofluorescent staining using antivon Willebrand factor antibodies (Ab), anti-{alpha}-SM-actin Ab, antivimentin Ab, and anti-human desmin Ab. The 3 endothelial-specific markers and the mesenchymal-specific marker were positive, and the 2 smooth muscle-specific markers were negative (Fig. 1 A), providing evidence that the commercially available HPMVEC were not contaminated with VSMC.


Figure 1
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  		Figure 1. Transdifferentiation of mature vascular endothelial cells to smooth muscle-like cells. A) HPMVEC were first confirmed to be free of contamination with vascular smooth muscle cells (VSMC) by staining for Ulex europaeus agglutinin-I, uptake of DiI-Ac-LDL, and immunofluorescent staining for antivon Willebrand factor, anti-{alpha}-SM-actin, Vimentin and Desmin. B) At 3 passages after SU5416 addition, HPMVEC were microscopically assessed. The magnification was 100x. C) HPMVEC were assessed by uptake of DiI-Ac-LDL and staining for Ulex europaeus agglutinin-I (Lectin). D) Positive cells for DiI-Ac-LDL and lectin were counted in 3 different fields at a magnification of 200x using a fluorescence microscope. Control (Lectin) vs. SU5416 (Lectin): *P < 0.05; n ≥ 3. E) HPMVEC treated with or without SU5416 were assessed by immunofluorescent staining for antivon Willebrand factor and anti-{alpha}-SM-actin to confirm transdifferentiation from EC to smooth muscle-like cells. Magnification was 200x. F) Positive cells for antivon Willebrand factor and anti-{alpha}-SM-actin were counted in 3 different fields at a magnification of 200x in a fluorescence microscope. *P < 0.05 vs. control, n ≥ 3.

At three passages after SU5416 addition, HPMVEC were assessed microscopically and morphological alterations were detected. The cell–cell contact of the endothelial monolayers became disrupted and many EC had lost their rounded appearance and acquired an elongated, mesenchymal-like morphology (Fig. 1B ).

There was no difference in the uptake of DiI-Ac-LDL and in the expression of vimentin and desmin between the cells treated with SU5416 and without SU5416 (Fig. 1C, D ), however, in SU5416-treated cells a decreased expression of Ulex europaeus agglutinin-I was detected (Fig. 1C, D ).

SM-like cells (as defined by expression of {alpha}-SM-actin) and transitional cells [coexpressing both endothelial- (von Willebrand factor) and SM- ({alpha}-SM-actin) cell markers] were consistently observed at 3 passages after SU5416 addition. The number of SM-like cells increased and the number of EC decreased at 5 passages after SU5416 addition (Fig. 1E, F ).

Treatment of HPMVEC with the VEGF antagonist SU5416 induces not only phenotypic but also genotypic alterations
We had previously identified decreased expression of PGI2S in the EC of the lungs from patients with severe PH (10) . Here we hypothesize that SU5416-treatment would reduce PGI2S expression. Using quantitative PCR, we found that indeed there was a decreased PGI2S gene expression in cells treated with SU5416 compared to cells without SU5416 (Fig. 2 A). While after 3 passages in vehicle-only treated cells PGI2S expression decreased, in the SU5416-treated cells PGI2S expression increased (Fig. 2A ).


Figure 2
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  		Figure 2. Gene expression of PGI2S, COX-2, VEGF, and TGF-ß1. Gene expressions of A) PGI2S, (B) COX-2, (C) VEGF, and (D) TGF-ß1 in EC were measured by quantitative RT-PCR. *P < 0.05 vs. passage 7, n ≥ 3.

An increase of COX-2, VEGF and TGF-ß1 gene expression in VEGF-R antagonist-treated cells was found when compared with vehicle-only treated (Fig. 2B-D ).

COX-2 and VEGF gene expression increased in the cells with or without SU5416 after 3 passages (Fig. 2B , C), while TGF-ß1 levels decreased in the cells not treated with SU5416 (Fig. 2D ).

Gene microarray analysis
Tables 1 and 2 list the differentially expressed genes in passage 7 HPMVEC 5 days after addition of the VEGF-R antagonist.


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  		Table 1. Functional classification of highly expressed genes in HPMVEC treated with SU5416 when compared with no SU5416


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  		Table 2. Functional classification of low expressed genes in HPMVEC treated with SU5416 when compared to no SU5416

The expression difference generated after VEGF-R blockade is characterized by up-regulation of a number of genes encoding proteins that belong to the TGF-ß signaling pathway; genes that control antiapoptotic proteins; the gene that encodes the 5-lipoxygenase activating protein (FLAP), which may be part of a cell survival program (31) ; 4 genes associated with muscle development: {alpha}2-actin, "capping protein", transgelin and tropomyosin 1{alpha}; and podocalyxin and EC differentiation gene EDG3. At 5 days after VEGF-R blockade, a large number of cell cycle control genes—among them cyclin A2, cyclin B1, and cell division cycle associated 1–3—are decreased in expression as are several genes encoding proteins involved in purine and pyrimidine metabolism, and arginase 2.

Transdifferentiation of CD34+ EC treated with SU5416, but not CD34– EC, to SM-like cells and neuronal-like cells
CD34 is a 105–120 kDa transmembrane glycoprotein expressed by hematopoietic progenitor cells, vascular EC, and some fibroblasts (36 37 38) . To test whether hematopoietic progenitor cells (as defined by CD34 expression) in the mature and normal HPMVEC transdifferentiate to mesenchymal-like cells, they were sorted for the marker CD34 and treated with or without SU5416.

After magnetic cell sorting for the marker CD34, HPMVEC were incubated with or without SU5416 and cultured until passage 12. At three passages after SU5416 addition, the cells were examined microscopically and morphological alterations were detected only in the CD34+ cells treated with SU5416. Cell-cell contact in endothelial monolayers became disrupted, and many EC had lost their rounded appearance and acquired an elongated, mesenchymal-like morphology (Fig. 3 A).


Figure 3
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  		Figure 3. Transdifferentiation from CD34+ EC treated with SU5416, not CD34– EC, to smooth muscle-like and mesenchymal-like cells. A) Light microscopy of CD34+ or CD34– HPMVEC at 3 passages after SU5416 addition. Magnification was 100x. B) Immunofluorescent staining of CD34+ or CD34– HPMVEC for von Willebrand factor and {alpha}-SM-actin to confirm SU5416-induced transdifferentiation of EC into smooth muscle-like cells. C) Light microscopy (a) and immunofluorescence staining for tyrosine hydroxylase (TH) (b) of CD34+ or CD34– HPMVEC at 5 passages after SU5416 addition. Magnification was 100x.

Immunofluorescent staining with antivon Willebrand factor Ab and anti-{alpha}-SM-actin Ab was used to assess SU5416-treated EC transdifferentiation. SM-like cells (as defined by expression of {alpha}-SM-actin) and transitional cells (coexpressing both endothelial and SM cells markers) were consistently observed in CD34+ cell populations at 3 passages after SU5416 addition and at passage 5 after SU5416 addition the number of SM-like cells further increased and number of EC decreased (Fig. 3B, F ).

At 5 passages after SU5416 addition a dendritic arbor-like morphology appeared in the CD34+ cell population (Fig. 3Ca ). To confirm the transdifferentiation of SU5416-treated EC into neuronal-like cells, the cells were assessed by immunofluorescent staining with tyrosine hydroxylase (TH) Ab. Neuronal-like cells (as defined by expression of TH) were consistently observed in CD34+ cell population at 5 passages after SU5416 addition (Fig. 3Cb ). Recently, it was reported by several groups that MSC can also adopt a neural fate in appropriate in vivo or in vitro experimental conditions (39 40 41 42) . However, it is unclear whether those cells and neuronal-like cells in our study can really differentiate into functional neural cells and in particular into functional neurons.

CD34 and c-kit expressions in HPMVEC
CD117 (c-kit) recognizes a 145 kDa cell-surface glycoprotein with tyrosine kinase activity. CD117 is present on hematopoietic progenitor cell subsets, thymocytes, mast cells, hepatocytes, and histiocytes (43 44 45 46) . To test whether the mature HPMVEC used in our experiments include hematopoietic progenitor cells, they were assessed by flow cytometry for both CD34 and/or c-kit. Increased expression of CD34 and c-kit was detected in HPMVEC at 3 passages after SU5416 treatment but not in the cells that had not been exposed to the VEGF-R antagonist (Fig. 4 ).


Figure 4
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  		Figure 4. CD34 and c-kit expressions in HPMVEC. HPMVEC treated with or without SU5416 was assessed by flow cytometry with CD34 and/or c-kit. *P < 0.05 vs. Passage 7, n ≥ 3.

Transdifferentiation of CD34+ c-kit+ is VEGF and TGF-ß1–independent
As shown above, SU5416 caused transdifferentiation from EC to SM-like cells. The gene expression of both VEGF and TGF-ß1 had increased in EC at 3 passages after SU5416 treatment as compared with vehicle-only treated EC. To assess whether the mediators that caused transdifferentiation were TGF-ß1 or VEGF, we incubated EC with SU5416 and neutralizing antibodies directed against TGF-ß1 (10 µg/ml) or VEGF (5 µg/ml) (47) and stained the cells with antivon Willebrand factor and anti-{alpha}-SM-actin Ab. Transdifferentiation of the SU5416 treated EC was not blocked by either VEGF or TGF-ß1 neutralizing antibodies (Figs. 5 and 1F ).


Figure 5
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  		Figure 5. VEGF and TGF-ß1 effect on transdifferentiation. Immunofluorescent staining for antivon Willebrand factor (green) and anti-{alpha}-SM-actin (red) of SU5416-treated HPMVEC with or without VEGF or TGF-ß1 neutralizing Ab. Transdifferentiation of the SU5416 treated EC was not blocked by either VEGF or TGF-ß1 neutralizing antibodies. Magnification 200x.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This series of experiments, which explored the life and death of lung microvascular EC, unexpectedly discovered the presence of a small number of bone marrow-derived c-kit+, CD34+ endothelial precursor cells among various batches of commercially available lung microvascular EC, suggesting the presence of such precursor cells in the adult lung. Furthermore, we demonstrate that these cells survive and expand in culture when exposed to the selection pressure of cell culture and VEGF receptor blockade and that these c-kit+, CD34+ cells are multipotent, since they give rise to VSMC-like cells and neuronal-like cells. The greater context of our findings may be a general mechanism for muscularization of vessels and in the nondeveloping adult lung a mechanism, which participates in lung tissue homeostasis and repair of injured lung cells via utilization of resident lung tissue precursor cells.

The prevailing theory of the VSMC contribution to vascular lesions is that in pathological states, like atherosclerosis, SMCs migrate to the intima from the media of the vessel (48) . This concept, however, has been challenged by results derived from models of vascular injury, transplant arteriosclerosis, and human allograft studies, which all indicate that a portion of the cells bearing SMC differentiation markers in intimal lesions may have originated from the hematopoietic system and/or circulating progenitor cells (49 50 51) . Furthermore, a recent study demonstrated that SM progenitors were present in circulating blood (52) , although the origin of these cells remains unknown. Concomitantly, it was shown that ~60% of SMCs in atherosclerotic lesions of vein grafts were derived from the donor vessel wall and 40% from the recipient, possibly from circulating blood cells (53 , 54) . In the aggregate these reports strongly suggest the possibility of stem or progenitor cells as the source of SM accumulation in atherosclerotic lesions. However, not all of the SMC within intimal lesions may be derived from bone marrow cells. Recently it was shown that, in addition to circulating progenitor cells, Sca-1+ progenitor cells that reside in the adventitia can transdifferentiate into SMC-like neointimal cells (20) , suggesting that not only bone marrow cells but also resident vessel wall precursor cells could exist and serve as a source of SMC to form neointimal lesions.

Our study demonstrates SU5416-induced transdifferentiation of HPMVEC to SM-like cells (Fig. 1A, B, E, F ). After VEGF blockade, the cells lost their EC features and acquired mesenchymal cell character. However, some EC features were conserved as shown by positive staining for Dil-ac-LDL of these cells (Fig. 1C, D ). We have also stained the transdifferentiated ECs with vimentin and desmin (Fig. 1C, D ). The transdifferentiated cells were {alpha}-SM actin- and vimentin-positive but desmin-negative. This finding indicates that these cells were transitional mesenchymal cells but not mature SM cells.

No transdifferentiation was found in the experiments of EC to mesenchymal-like cells to indicate that HPMVECs were induced to undergo apoptosis by applying human TNF-{alpha} (10 ng/ml) and cycloheximide (10 µg/ml) for 24 h or hydrogen peroxide (H2O2) (1 mmol/L) for 16 h to the cells (data not shown). These experiments suggest that apoptosis itself is not sufficient to induce transdifferentiation and that VEGF is a key mediator in this process. Recent study by Rufaihah et al. (55) demonstrated that transduction of differentiating human embryonic stem cells (hESCs) with an adenoviral vector expressing the VEGF (165) gene can enhance endothelial-lineage differentiation. While here we show that loss of VEGF and VEGF signaling promotes growth of smooth muscle-like and neuronal-like cell.

The observed decrease in PGI2S gene expression in HPMVEC after VEGF-R blockade (Fig. 2A ) is another indication of an altered EC phenotype. This finding is consistent with the loss of PGI2S expression in EC of the lungs from patients with severe PH (10) .

The common diagnostic endothelial marker CD34 is a 115 kDa transmembrane glycoprotein, present on lymphohematopoietic stem and progenitor cells, leukemic cells, EC, and embryonic fibroblasts. In clinical practice CD34 is used as a marker for leukemia diagnosis and subclassification, as a label for the quantity of stem/progenitor cells in blood and marrow and also for purification of stem/progenitor cells for clinical transplantation (56 , 57) . CD34 is up-regulated in vivo during angiogenesis (58) and is primarily expressed by small or newly formed vessels (59) , whereas EC of larger veins, placenta, and lymphatic tissue are CD34-negative (59) . CD34 was reported to be expressed in alveolar capillaries rather than in large pulmonary vessels (60) . We interpret our data in the following way: VEGF blockade generated a selection pressure for the expansion of an originally small number of CD34-positive cells to proliferate and differentiate (Fig. 3A-C ). This finding indicates the presence of some resident vascular precursor cells (as defined by CD34 expression) in commercially available lung microvascular EC and implies the presence of such precursor cells in the adult lung. Muller et al. (60) recently demonstrated that 20% of EC in human umbilical vein endothelial cells (HUVEC) and HPMVEC (passage 3) are CD34-positive. In comparison, we found that only around 2–3% of HPMVEC (passage 7) were CD34 positive (Fig. 4) ; this discrepancy may be passage-dependent.

VEGF blockade also caused expansion of c-kit positive cells (Fig. 4) in addition to CD34+ cells, supporting the interpretation that VEGF blockade expanded a progenitor cell pool, since c-kit, like CD34, is expressed in bone marrow-derived hematopoietic progenitor cells (45) .

Overexpression of COX-2 has been identified early in carcinogenesis, and up-regulation of COX-2 has been described in lung cancer (29) . Because we have previously demonstrated that EC death induced by SU5416 results in the selection of an apoptosis-resistant, proliferating, and phenotypically altered EC phenotype (30) , here we wondered whether in EC treated with SU5416 the COX-2 gene expression was increased. Indeed, not only COX-2, but also VEGF and TGF-ß1 gene expression, were increased in the cells treated with the VEGF-R antagonist when compared with cells not treated with SU5416 (Fig. 2B-D ).

In our experiments, the SU5416-induced transdifferentiation of HPMVEC into mesenchymal-like cells was independent of VEGF or TGF-ß1 (Figs. 5 and 1F) . Although TGF-ß1 was shown to be involved in inducing endothelial-mesenchymal transdifferentiation (17) and is known to promote {alpha}-SM-actin expression in nonmuscle cells (EC and fibroblasts derived from various tissues) (19 , 61) , TGF-ß1 is currently thought to be insufficient to induce expression of late SM differentiation marker SM myosin heavy chain (SM-MHC) in non-SMC lineage cells (61) . Thus, it is possible that TGF-ß1 is necessary for initiation of transdifferentiation, yet it may not be sufficient for driving the process to a higher level of SM differentiation as defined by the expression of SM-MHC.

The outcome of our experiments, i.e., the particular cell fate and phenotype may depend on the initiating events, i.e., VEGF receptor blockade, EC apoptosis and the multitude of signals generated in the environment of dying cells, which act on the surviving CD34+ and c-kit+ cells.

This milieu also permits the appearance of multinucleated and neuronal-like, tyrosine hydroxylase-positive cells (Fig. 6 ).


Figure 6
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  		Figure 6. Sequence of events in HPMVEC that lead from VEGF blockade by SU5416 to transdifferentiation to smooth muscle-like cells. Endothelial cell death induced by VEGF receptor blockade and subsequent selection of progenitor-like cells leads to transdifferentiation to smooth muscle-like cells.


   ACKNOWLEDGMENTS
 
The work was supported by NIH 5P01 HL66254–03 PI and a NIH Program Project Grant (NFV). The authors thank Dr. John M. Stewart, Department of Biochemistry, University of Colorado at Denver and Health Sciences Center for the critical reading of this manuscript. This work is dedicated to the memory of Dr. J. T. Reeves.

Received for publication February 27, 2007. Accepted for publication May 17, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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