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Am J Physiol Lung Cell Mol Physiol 294: L431-L439, 2008. First published November 2, 2007; doi:10.1152/ajplung.00316.2007
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EDITORIAL FOCUS

Regulatory role for nucleosome assembly protein-1 in the proliferative and vasculogenic phenotype of pulmonary endothelium

Jennifer Clark,1,4 Diego F. Alvarez,4 Mikhail Alexeyev,2,4 Judy A. C. King,1,3,4 Lan Huang,5 Mervin C. Yoder,5,6 and Troy Stevens1,4

Departments of 1Pharmacology, 2Cell Biology and Neuroscience, and 3Pathology, 4Center for Lung Biology, University of South Alabama College of Medicine, Mobile, Alabama; and Departments of 5Biochemistry and Molecular Biology and 6Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana

Submitted 6 August 2007 ; accepted in final form 31 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary microvascular endothelial cells (PMVECs) are enriched with progenitor cells that underlie their rapid proliferation and vasculogenic capacity. However, the molecular basis for such an enhanced growth potential is unknown. Nucleosome assembly protein-1 (NAP1), and its related family of proteins, have been incriminated in control of cell growth in a range of species. We therefore sought to determine whether NAP1 contributes to the rapid proliferation and vasculogenesis that is observed in PMVECs. NAP1 was expressed at a high level in two fast-growing cell types, including PMVECs and resident microvascular endothelial progenitor cells that were selected from PMVECs, whereas it was expressed at a low level in slow-growing pulmonary artery endothelial cells (PAECs). Heterologous NAP1 expression increased the growth potential of PAECs, whereas inhibiting NAP1 expression reduced the growth potential of PMVECs. Despite its impact on endothelial cell growth, NAP1 did not influence the expression of endothelial cell-selective markers (PECAM-1, VE-cadherin, von Willebrand factor), and it did not influence cell type-specific lectin binding criterion; PMVECs interact with Griffonia simplicifolia lectin, whereas PAECs interact with Helix pomatia lectin. PMVECs possess a higher basal transelectrical resistance than do PAECs, indicative of their more restrictive barrier property. Changing NAP1 expression did not normalize this basal barrier function between PMVECs and PAECs. To examine whether the growth-promoting actions of NAP1 influence blood vessel formation, endothelial cells were mixed into Matrigel and subcutaneously implanted. PMVECs generated eightfold more blood vessels than did PAECs over a 10-day time course. Heterologous NAP1 expression in PAECs increased the number of blood vessels formed by this cell type, where blood vessel growth approached that seen with PMVECs. Thus, our findings indicate that NAP1 functions as an important regulator of the proliferative and vasculogenic endothelial cell phenotype without globally impacting endothelial cell phenotype specification.

angiogenesis; microcirculation; endothelial cells; pulmonary vasculature

ENDOTHELIUM LINES ALL BLOOD and lymphatic vessels and separates blood and lymph from the underlying tissue (1, 2, 10). Routine cell turnover is necessary to renew aged and injured endothelium, to ensure cardiovascular health. Estimates from conduit vessels, such as the aorta, suggest that endothelium is renewed every 1–3 years (1, 6). Microvascular endothelial cell turnover has not been similarly measured, although it is evident that pulmonary microvascular endothelial cells (PMVECs) grow at a faster rate, both in vivo and in vitro, than do their macrovascular counterparts (4, 17, 27). Such an enhanced proliferative potential is due to the extraordinary number of progenitor cells that reside within PMVEC populations (4). These resident microvascular endothelial progenitor cells not only account for the rapid proliferation of microvascular endothelial cells but also underlie their enhanced vasculogenic potential. Indeed, when mixed in Matrigel and subcutaneously implanted, PMVECs form more blood vessels than do pulmonary artery endothelial cells (PAECs) (4). While the proliferative and angio/vasculogenic nature of PMVECs has been resolved, mechanistic insight into these phenomena is still lacking.

Lung microvascular endothelial cells possess increased expression and activity of proliferative factors compared with PAECs (27). High PMVEC proliferation is accompanied by increased cyclin D1 protein expression, and increased cyclin-dependent kinase complex activity results in retinoblastoma hyperphosphorylation. Serum restriction is not sufficient to completely arrest PMVECs in G0/G1, although cell-cell contact at confluence is sufficient to arrest PMVEC growth. These findings suggest that PMVECs possess an intrinsic proliferative imprint that accounts for their rapid growth following barrier disruption (inhibition of cell-cell contact).

We sought to identify a factor(s) that may coordinate cell growth and differentiation and contribute to the stable proliferative PMVEC imprint. Nucleosome assembly protein-1 (NAP1), and the related NAP1 family, has recently been incriminated in control of proliferative phenotype, most notably in yeast, Xenopus, and Arabidopsis thaliana (3, 7, 9, 18–20, 24, 26, 29, 31). In Arabidopsis, NAP1 was necessary for cell proliferation in leaf development (9), whereas NAP1-related proteins 1 and 2 were required for root growth (31). NAP1 likely regulates cell proliferation by interacting with cyclin B, where it promotes proper assembly of the mitotic spindle (3), and by interacting with histones 2A and 2B, where it chaperones histones into the nucleus and dynamically controls their assembly into nucleosomes that control transcriptional activation (8, 13, 14, 22). In addition to controlling chromatin assembly, NAP1 and related proteins appear to specifically influence the expression of growth and apoptotic genes, suggesting NAP1 plays a central role in epigenetic inheritance of proliferative traits (19, 31). NAP1 knockout mice have not been generated, and little information is available in mammalian systems to determine whether NAP1 controls the proliferative phenotype. However, inactivation of the NAP1-like 2 gene in mice results in an early gestation (embryonic day 10.5) neural tube defect that is likely due to overproduction of nestin-positive precursor cells (24). In Drosophila melanogaster, NAP1 gene deletion similarly causes early embryonic lethality, although the underlying mechanism has not been determined (18). At present, it is not clear whether NAP1 has any role in endothelial cell growth, and, more specifically, in angio-/vasculogenesis. To address this issue, we examined whether proliferating endothelial cells express NAP1 and whether NAP1 abundance is a critical determinant of endothelial cell vasculogenic potential.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Western blot analysis. NAP1 protein was resolved using standard Western blotting, as described elsewhere (13, 14). Endothelial cells were grown to confluence in 100-mm culture plates treated with whole cell lysis buffer (150 mmol/l NaCl, 10 mmol/l Tris·HCl, pH 7.2, 5 mmol/l MgCl2, 2 mmol/l EDTA, 0.25 mmol/l DTT, 1 mmol/l PMSF, 1% Triton X-100, supplemented with 1 mmol/l protease inhibitor cocktail) and centrifuged (3,000 g, 15 min, 4°C). For nuclear protein extraction, 150 mmol/l dishes were cultured to confluence, placed on ice, and rinsed with cold PBS three times. Cells were then scraped into 5 ml of cold PBS and centrifuged (2,000 g, 10 min, 4°C). Cells were then washed with 5 packed cell volume of 10 mmol/l Tris·HCl, pH 7.5, 1.5 mmol/l MgCl2, 10 mmol/l KCl, 1 mmol/l protease inhibitor cocktail, and 1 M DTT, resuspended in 4 packed cell volume of the same buffer and incubated on ice for 10 min. Cells were then homogenized in a glass Dounce homogenizer using a loose pestle with 15 strokes and centrifuged (10,000 g, 10 min, 4°C). Pelleted nuclei were resuspended in 20 mmol/l Tris·HCl, pH 7.5, 20 mmol/l KCl, 1.5 mmol/l MgCl2, 20% glycerol, 1 mmol/l protease inhibitor cocktail, 1 mmol/l DTT, rotated for 30 min at 4°C, and centrifuged (15,000 g, 30 min, 4°C). The supernatant was then dialyzed against two changes of 25 mmol/l Tris·HCl, 0.2 mmol/l EDTA, 100 mmol/l KCl, 20% glycerol, 1 mmol/l DTT, and 1 mmol/l protease inhibitor cocktail for 4 h at 4°C. Protein concentration was determined using Bradford protein assay kit (BioRad) for SDS-PAGE using 7% Tris-acetate gels (Invitrogen, Carlsbad, CA). The proteins were transferred to nitrocellulose membrane at 30 V for 2 h. Membranes were rocked overnight at 4°C with primary antibody to NAP1 (Dr. Ishimi, Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan; cl.4A8) and with horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature. Proteins were visualized via enhanced chemiluminescence detection (SuperSignal West Pico Chemiluminescent Substrate, Pierce) and developed using Hyperfilm ECL (Amersham Biosciences) and a Hope micromax film processor.

Retroviral infection. PAECs were seeded at 100,000/well in a six-well plate and cultured for 48 h in the presence of DMEM (10% FBS, 1% penicillin/streptomycin, standard culture media) at 37°C in a humidified incubator. Media was removed, and 0.5 ml of culture supernatant containing retroviral construct generated in our gene delivery core supplemented with polybrene (4 µg/ml) was added. Plates were centrifuged at 750 g for 30 min at 37°C. Plates were returned to the incubator for 24 h after which retroviral culture supernatant was replaced with standard culture media. After 48 h, successfully infected cells were selected using Hygromycin (50 µg/ml). PMVECs were infected with short hairpin RNA (shRNA) containing constructs as above, and successfully infected cells were selected using puromycin (20 µg/ml).

Confocal microscopy. NAP1 expressing PAECs (NAP1 PAECs) were seeded (100,000 cells) on glass coverslips and cultured for several days in the presence of standard culture media. Cells were fixed in 100% methanol, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 2% serum in PBS. Mouse primary antibody to hemagglutinin (HA; Sigma; 1:1,000 in PBS containing 2% serum) was incubated for 1 h at room temperature, followed by Alexa Fluor 488-conjugated secondary antibody (rabbit anti-mouse) for 30 min at room temperature. Nuclei were labeled using Draq5 red fluorescent cell-permeable DNA probe (Biostatus). Fluorescence staining was visualized using an Ultraview RS-3 Spinning-disk Confocal System (PerkinElmer).

Proliferation analysis. PAECs, PMVECs, NAP1 PAECs, and shRNA-244 PMVECs were seeded in multiple six-well plates at 100,000 cells/well in the presence of standard culture media. Growth was determined by cell counts every 24 h using a Beckman Coulter Counter. Growth curves were generated using GraphPad Prism and analyzed using one-way ANOVA.

Cell cycle analysis. PAECs, PMVECs, and NAP1 PAECs were seeded at 4 x 104 cells/well in multiple six-well plates. After 24 h of growth in standard culture media (10% serum), media was changed to contain 0.1% serum for 72 h to induce growth arrest. Following growth arrest, serum-deprived media was replaced with standard culture media. Cell cycle profiles were then determined every 24 h using fluorescence-activated cell sorting (FACS) analysis following propidium iodide staining. Data were analyzed using GraphPad Prism and two-way ANOVA.

FACS. PAECs, PMVECs, NAP1 PAECs, and shRNA-244 PMVECs were grown to confluence in T-75 flasks, trypsinized, filtered, centrifuged, and resuspended in PBS. Cells were permeabilized using 0.1% Triton X-100 in PBS. Cells (106) were loaded in 1.7-ml centrifuge tubes and incubated with primary antibody (1:50 in PBS) to VE-cadherin, von Willebrand factor, and PECAM for 30 min on ice. Cells were then rinsed with PBS and incubated with FITC-conjugated secondary antibody for 30 min on ice, followed by rinsing. The FITC-conjugated Griffonia simplicifolia and Helix pomatia lectins were incubated with cells for 30 min on ice, followed by rinsing. As a control, cells were incubated with only the secondary antibody or IgG isotype FITC. Following fluorescent labeling, cells were resuspended in PBS and analyzed by flow cytometry.

Electrical cell-substrate impedance sensing. PAECs, PMVECs, NAP1 PAECs, and shRNA-244 PMVECs were seeded at equivalent densities (4 x 104 per well) on gold electrodes (ECIS Cultureware 8W10E, Applied BioPhysics) in 400 µl of standard culture media. Media was changed after 4 days and every day thereafter. Resistance measurements were recorded daily for comparison of barrier properties during monolayer formation using Electrical Cell-Substrate Impedance Sensing morphological biosensor Model 1600R (Applied BioPhysics). Data were analyzed using GraphPad Prism (1-way ANOVA).

Matrigel in vivo. Cells (3.75 x 105) were mixed with 500 µl of unpolymerized Matrigel (BD laboratories) and 250 µl of standard culture media at 4°C to maintain the mixture in a fluid phase. Matrigel mixture was injected subcutaneously into the abdomen of mildly sedated (ketamine 75 mg/kg ip) 10- to 15-wk-old (250–300 g) CD40 male rats (Charles River Laboratories). The injected mixture solution polymerized at body temperature following subcutaneous contact (typically <1 min). In control experiments, Matrigel was injected (500 µl) with no cells. Rats were euthanized 10 days postinjection using a barbiturate overdose followed by exsanguination via cutting of the right ventricle. Matrigel plugs were excised from the abdominal wall and immersion fixed in 4% paraformaldehyde. Fixed specimens were embedded in paraffin and cut into 5-µm sections. Sections were stained with hematoxylin and eosin for examination by light microscopy. Sections were examined to count the total number of tubes containing red blood cells and analyzed using GraphPad Prism (2-way ANOVA). Animal studies were approved by the Institutional Animal Care and Use Committee at the University of South Alabama.

Immunohistochemistry. Matrigel plugs immersion fixed in 4% paraformaldehyde were embedded in paraffin and cut into 5-µm sections. Sections were deparaffinized with two washes of xylene for 10 min each. Tissue was then rehydrated using a gradual decrease in alcohol (100%, 70%, 50%, and 30%) content in 5-min time intervals. Slides were immersed in water for 10 min. Tissues were permeabilized using 0.15% Triton X-100 in PBS. Tissue was heated (80°C) in the presence of 10 mmol/l sodium citrate, pH 6.0, for 20 min. Slides were allowed to cool and were rinsed in PBS three times for 5 min. Tissue sections were incubated with blocking buffer (2% serum, 4% BSA, 0.5% milk protein in PBS) for 30 min at room temperature. Blocking buffer was removed, and primary antibody (1:200 in PBS, 2% serum, 4% BSA) to HA (Abcam) was incubated overnight at 4°C. Slides were then rinsed in PBS three times for 5 min each followed by 30 min in the presence of horseradish peroxidase-conjugated secondary antibody (1:1,000 in PBS, 2% serum) goat anti-mouse (Sigma). Slides were rinsed three times for 5 min in PBS. Tissue sections were incubated in 3% hydrogen peroxide for 10 min followed by three more PBS washes of 5 min each. Slides were developed using Dako Liquid DAB Substrate-Chromagen System, and slides were counterstained with hematoxylin (2 min) and rinsed in water (5 min).

Transmission electron microscopy from in vivo studies. Matrigel plugs were excised after 10 days postinjection and immersion-fixed in 3% glutaraldehyde (in cacodylate buffer). Specimens were processed for transmission electron microscopy and examined as described previously (4).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Endothelial cells express NAP1. PMVECs proliferate faster than do PAECs. Cyclin D1 expression is threefold higher in PMVECs than in PAECs, and the microvascular cells also possess greater cdk4 and cdk2 activity, resulting in hyperphosphorylation and inactivation of retinoblastoma (27). Faster PMVEC growth is due to an abundance of highly proliferative progenitor cells that reside within the PMVEC population (4). However, the molecular basis for inducing and sustaining such a proliferative behavior remains uncertain. We therefore reviewed mRNA profiling results comparing PMVECs and PAECs to determine whether chromatin-associated genes that may influence growth potential were differentially expressed among these cell types. Review of mRNA profiling data revealed a 3.5-fold greater expression level of NAP1 in PMVECs than in PAECs (data not shown). NAP1 was an intriguing candidate gene for control of a proliferative phenotype in PMVECs, as prior studies demonstrated that nucleosome assembly proteins play an essential role in stem cell and hematopoietic progenitor cell differentiation (29).

We initially sought to confirm that the increased NAP1 mRNA detected in PMVECs resulted in increased protein abundance. Western analysis of whole cell lysates revealed two distinct NAP1 bands, one at ~58 kDa and one at ~53 kDa (Fig. 1A). These results are in good agreement with previous studies using MAb targeting the 4a8 protein epitope; the lower band is thought to reflect a NAP1 degradation product (13, 14). PMVECs possessed greater abundance of both the 58- and 53-kDa NAP1 than did PAECs. In interphase cells, NAP1 is resolved in both the cytosol and the nucleus. In the cytosol, it binds to histones 2A and 2B and is responsible for chaperoning these proteins into the nucleus. In the nucleus, NAP1 dynamically interacts with histones and p300 to coordinate gene expression (5, 15, 25). We therefore examined whether NAP1 was present in the nuclear fractions of quiescent PMVECs and PAECs. Whereas both 58- and 53-kDa bands were present in PMVEC nuclear fractions at high abundance, only the larger band was readily detectable in PAEC nuclear fractions (Fig. 1B). Thus, PMVECs constitutively possess greater amounts of NAP1 in both cytosolic and nuclear fractions than do PAECs.


Figure 1
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  		Fig. 1. Pulmonary microvascular endothelial cells (PMVECs) express more nucleosome assembly protein-1 (NAP1) than do pulmonary artery endothelial cells (PAECs). Western analysis from confluent monolayers of PMVECs and PAECs reveals greater NAP1 levels in PMVECs, both in whole cell lysates (A) and nuclear fractions (B). C: resident microvascular endothelial progenitor cells (RMEPCs) express high levels of NAP1 in whole cell lysates. MW, molecular weight.

 
We next sought to determine whether high NAP1 expression is common among fast-growing endothelial cells. PMVEC populations in culture are enriched with fast-growing progenitor cells that can be selected by single cell cloning (4, 11, 12). In a single cell clonogenic assay, PMVECs are seeded at single cell density and grown in 10% serum for 2 wk (4). Fully differentiated cells remain as single cells; only ~25% of the PMVECs previously studied remained as single cells. Nearly 75% of PMVECs grow into colonies, with 50% of the cells giving rise to large colonies of more than 2,000 cells. Replating the cells that grew to more than 2,000 cells at single cell density renews the entire hierarchy of fully differentiated, slow- and fast-growing cells. Based on their high proliferation rate and the ability to self-renew the PMVEC colony, fast-growing cells fulfill the criterion for progenitor cells (resident microvascular endothelial progenitor cells, RMEPC). We therefore tested RMEPCs to determine whether they express high NAP1 levels (Fig. 1C). RMEPCs possess high NAP1 abundance, consistent with the idea that high NAP1 expression contributes to the endothelial growth potential.

NAP1 promotes endothelial cell proliferation. To determine more directly whether NAP1 contributes to the proliferative phenotype of endothelium, NAP1 expression was increased in PAECs using retroviral infection of plasmid containing HA tagged to full-length NAP1, driven with a cytomegalovirus promoter (Fig. 2A). The plasmid contained a hygromycin resistance cassette, allowing NAP1-expressing cells to be selected to homogeneity. Immunocytochemical analysis confirmed expression of the HA-tagged NAP1 construct in PAECs (Fig. 2B). HA-tagged NAP1 was detected in both the cytosol and the nucleus of interphase cells. In contrast, HA-tagged NAP1 was localized exclusively in the nucleus of dividing cells (data not shown). Western analysis revealed the HA-tagged protein resolves at ~58 kDa, similar to endogenous NAP1 (Fig. 2C). NAP1 overexpression in PAECs increases total protein abundance to levels that resemble the endogenous NAP1 expression seen in PMVECs (Fig. 2D). Thus, HA-tagged NAP1 cellular localization resembles that of endogenous NAP1, providing the opportunity to determine whether increased NAP1 induces a proliferative phenotype in endothelium.


Figure 2
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  		Fig. 2. NAP1 was expressed in PAECs using a retroviral construct. A: schematic of the retroviral construct for hemagglutinin (HA)-tagged NAP1 expression in PAECs, containing hygromycin resistance cassette. B: PAECs were infected with retrovirus expressing NAP1 and selected to homogeneity using hygromycin. Immunocytochemistry (confocal microscopy) using primary HA antibody, secondary Alexa Fluor 488, and DRAQ for nuclear stain (blue) reveals HA-NAP1 expression (green) in the cytosol and nucleus (x60). Arrow denotes nuclear HA-NAP1. C: Western analysis of whole cell lysates following hygromycin selection using antibody to HA reveals construct expression corresponding to proper 58-kDa size. D: Western analysis using whole cell lysates reveals that NAP1 overexpressing PAECs and wild-type (nontransfected) PMVECs possess similar NAP1 abundance.

 
To measure cell growth, PAECs and PMVECs were seeded at subconfluence, cell cycle was synchronized using 0.1% serum for 2 days, and serum (10%)-stimulated growth was documented over a 6-day (144 h) time period. As in our earlier reports (4, 17, 27), PMVECs grew faster than did PAECs (Fig. 3A). NAP1 overexpression increased the growth rate of PAECs to levels resembling that seen with PMVECs (Fig. 3B) (PMVECs: 27 x 105 cells in 144 h; NAP1 PAECs: 28 x 105 cells in 144 h). Cell cycle analysis revealed that nearly 40% of PMVECs were in the S-phase of the cell cycle at subconfluence compared with just 25% of PAECs (Fig. 3C). NAP1 overexpression increased the number of PAECs in S-phase to nearly 35% at subconfluence. Confluence dramatically decreased the number of cells in S-phase to between 5 and 15% in PMVECs, PAECs, and NAP1-expressing PAECs. Thus, stably increasing NAP1 expression is sufficient to increase PAEC proliferation, to achieve growth rates resembling that seen in PMVECs, while retaining cell-cell contact inhibition.


Figure 3
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  		Fig. 3. Increased NAP1 in PAECs confers a proliferative/cell cycle profile similar to PMVECs in vitro. A: PMVECs proliferate faster in culture compared with PAECs. Cells were seeded at equivalent densities, grown in 10% FBS DMEM, and counted daily for 6 days (144 h). Data were analyzed using GraphPad Prism, 2-way ANOVA. *P < 0.01 vs. PAEC. B: PAECs were infected with retrovirus expressing NAP1 or with retrovirus expressing hygromycin resistance cassette lacking NAP1 (control PAEC). NAP1 expression in PAECs increases their growth. *P < 0.01 vs. control PAEC. C: cell cycle analysis using propidium iodide staining reveals NAP1 overexpression significantly increases the S-phase population throughout growth to confluence compared with PAECs. *P < 0.01 vs. NAP1 PAEC.

 
We next sought to determine whether a shRNA approach could be adapted to stably reduce NAP1 levels in PMVECs and inhibit their proliferation. NAP1 mRNA encoding the full-length protein is 2,122 nucleotides. Four separate shRNA constructs were generated to target discrete regions of the NAP1 mRNA, including 181-201 (shRNA-181), 224-244 (shRNA-224), 409-429 (shRNA-409), and 412-432 (shRNA-412) (Fig. 4A). These shRNA constructs were inserted into retrovirus containing a puromycin-resistant cassette, and expression was driven using an H1 promoter. PMVECs were infected with retrovirus, and cells expressing shRNA were selected to homogeneity. Selected cells were then grown to confluence, and NAP1 abundance was evaluated in cytosolic and nuclear fractions. NAP1 abundance was decreased in both fractions of interphase cells expressing shRNA-224 (Fig. 4B), but not in cells expressing shRNA-181 (data not shown), shRNA-409, and shRNA-412 (data not shown). We therefore performed population growth studies using cells expressing shRNA-224 and shRNA-409 as a control. shRNA-409-expressing cells grew to confluence with ~35 x 105 cells over a 6-day time course; this growth rate was similar to that observed in nontransfected PMVECs (see Fig. 3A). However, shRNA-224-expressing cells grew to confluence with ~12 x 105 cells over a 6-day time course; this growth rate was similar to that observed in wild-type PAECs (see Fig. 3A). Thus, shRNA-224 reduced NAP1 protein and also inhibited PMVEC growth.


Figure 4
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  		Fig. 4. Inhibiting NAP1 expression in PMVECs decreases proliferation. A: short hairpin RNA (shRNA) constructs were generated to target 4 different portions of the NAP1 message. B: shRNA constructs were inserted in retrovirus for expression. PAECs were infected with retrovirus expressing shRNA and selected to homogeneity using puromycin. shRNA-224 reduced NAP1 levels in PMVEC whole cell lysates and nuclear extracts, whereas shRNA-409 was without effect. C: using shRNA-409 as a retroviral control, growth curve analysis revealed decreased NAP1 levels in PMVECs (shRNA-224) resulted in a growth curve similar to that observed in PAECs, whereas growth was unaltered in cells expressing shRNA-409. *P < 0.01 vs. shRNA-409.

 
NAP1 does not change the global endothelial cell phenotype. We next examined whether NAP1 critically regulates the endothelial cell phenotype, and, more specifically, whether NAP1 changes macrovascular vs. microvascular endothelial cell specification. Both PAECs (Fig. 5A) and PMVECs (Fig. 5C) express classic endothelial cell markers PECAM-1, VE-cadherin, and von Willebrand factor. Neither NAP1 overexpression in PAECs (Fig. 5B) nor NAP1 inhibition in PMVECs (Fig. 5D) changed this classic marker pattern. Whereas PAECs interact with Helix pomatia lectin, PMVECs interact with Griffonia simplicifolia lectin, both in vivo and in vitro (17). This lectin-binding pattern was retained irrespective of NAP1 expression (Fig. 5). Thus, whereas NAP1 expression is an important determinant of the cell growth potential, it does not change endothelial cell phenotype specification.


Figure 5
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  		Fig. 5. NAP1 does not alter endothelial cell specification. A: FACS analysis of PAECs reveal they express PECAM-1, VE-cadherin, and von Willebrand factor (vWfactor). PAECs interact with Helix pomatia lectin and do not interact with Griffonia simplicifolia lectin. B: NAP1 overexpressing PAECs similarly express PECAM-1, VE-cadherin, and vWfactor, and interact with Helix pomatia lectin. C: PMVECs express PECAM-1, VE-cadherin, and vWfactor, and interact with Griffonia simplicifolia, but not Helix pomatia, lectin. D: shRNA-224 expressing PMVECs possess PECAM-1, VE-cadherin, and vWfactor, and interact with Griffonia simplicifolia lectin. E: basal transelectrical membrane resistance is higher in PMVECs than in PAECs. Membrane resistance was increased in NAP1 overexpressing PAECs, but NAP1 expression did not normalize the resistance in PAECs to levels observed in PMVECs. Membrane resistance was decreased in shRNA expressing PMVECs, but decreased NAP1 expression did not normalize resistance in PMVECs to levels observed in PAECs. *P < 0.01, #P < 0.05, as analyzed using GraphPad Prism 1-way ANOVA.

 
To gain further insight into whether NAP1 regulates other functional attributes of endothelium, we determined whether the NAP1 expression pattern changes adhesion strength. PMVECs form a tighter barrier than do PAECs, both in vivo and in vitro (16, 23, 28). Basal transelectrical membrane resistance (TER) was measured to assess barrier function. As previously reported (4), PMVECs possessed a higher basal resistance than did PAECs (Fig. 5E). Decreasing NAP1 expression in PMVECs slightly reduced basal TER, but not to the level observed in PAECs. Increasing NAP1 expression in PAECs slightly increased TER, but not to the level observed in PMVECs. Thus, NAP1 abundance does not globally change PAEC or PMVEC adhesion strength.

NAP1 promotes blood vessel formation. PMVECs are rapidly neovasculogenic in in vivo Matrigel plug assays (4). We therefore sought to determine whether NAP1 expression regulates endothelial cell vasculogenic capacity. PAECs and PMVECs were mixed with Matrigel and subcutaneously injected in the abdomen. Our prior studies resolved a greater number of new blood vessels formed in PMVEC-seeded plugs than in PAEC-seeded plugs 10 days after Matrigel implantation (4). We therefore quantitated vessel formation after 10 days and confirmed that plugs seeded with PMVECs generate a greater number of new blood vessels than do PAECs (Fig . 6, A and I). We investigated whether NAP1 overexpression increases the vasculogenic capacity of PAECs. To perform this study, PAECs were infected with retrovirus-expressing NAP1, and the cells were selected to homogeneity using hygromycin and then grown to confluence. The NAP1-overexpressing cells were seeded for a proliferation study and mixed with Matrigel for subcutaneous injection. We noted that cell growth was increased over the entire 10-day time course in NAP1-overexpressing cells (Fig. 6B). Vessel counts also revealed that NAP1 overexpression increased the number of blood vessels documented within the plug (Fig. 6, C and I), indicating that NAP1 expression promotes vasculogenesis. TEM was performed to assess the quality of vessels formed in NAP1-overexpressing cells. Figure 6D illustrates that NAP1-overexpressing PAECs seeded in Matrigel form normal vascular structures, with functional lumens and electron dense complexes at cell-cell junctions. To confirm that cells injected in the plugs were responsible for lumen formation, PAECs expressing the HA-NAP1 construct were detected using an HA antibody. As seen in Fig. 6E, HA-NAP-positive staining was found in cells lining the lumen of newly formed vessels, indicating that injected cells, and not cells recruited from the animal, were responsible for blood vessel formation.


Figure 6
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  		Fig. 6. NAP1 overexpression increases blood vessel formation in Matrigel plugs in vivo. A: light micrograph of PMVEC-seeded Matrigel plug removed after 10 days and stained with hematoxylin and eosin reveals vessel formation (x40). Arrow indicates vessel containing red blood cells (RBC). B: growth curve analysis of NAP1 overexpressing PAECs over a 10-day (240 h) time course illustrates fast growth rates are evident for the entire time course. C: light micrograph of NAP1 PAEC-seeded Matrigel plugs removed after 10 days and stained with hematoxylin and eosin reveals dense vessel formation (x40). Arrow indicates vessel containing RBC. D: transmission electron micrograph of Matrigel plugs seeded with NAP1 overexpressing PAECs. Plugs removed after 10 days reveal the NAP1 overexpressing cells form normal blood vessels with RBC in the lumen (left). Electron dense adhesion sites are prominent at cell-cell borders (arrows; right). EC, endothelial cell. E: light micrograph of immunohistochemical analysis of HA-tagged, NAP1-expressing PAECs embedded in Matrigel plugs illustrates injected cells form the lumen of new blood vessels (x60). F: growth curve analysis of shRNA-224-expressing PMVECs reveals loss of growth inhibition between days 7 and 10. G: Western analysis of whole cell lysates obtained from shRNA-224-expressing PMVECs reveals NAP1 abundance has rebounded to levels observed in NAP1-overexpressing PAECs. H: light micrograph of shRNA-224-expressing PMVEC-seeded Matrigel plugs removed after 10 days and stained with hematoxylin and eosin (x40). Arrow indicates vessel containing RBC. I: vessel density assessment reveals PMVECs form significantly greater numbers of vessels than do PAECs (P < 0.01). NAP1 overexpression significantly increases vessel density (P < 0.01). *P < 0.01 as analyzed by GraphPad Prism 1-way ANOVA.

 
We next attempted to determine whether inhibition of NAP1 expression in PMVECs is sufficient to reduce vasculogenesis. PMVECs were infected with retrovirus-expressing shRNA-224, selected using puromycin, and grown to confluence in the absence of antibiotic. shRNA-224-expressing cells were then seeded for cell growth studies and mixed into Matrigel for subcutaneous injection. We noted that while shRNA-224 reduced PMVEC growth for 7 days, as reported in Fig. 4C, growth inhibition was lost between days 7 and 10 in culture (Fig. 6F). Western analysis of whole cell and nuclear fractions revealed that by day 10, shRNA-224 no longer reduced NAP1 expression; indeed, by 10 days, NAP1 expression in shRNA-224-treated PMVECs was higher than it was in untreated cells and resembled abundances observed in RMEPCs (Fig. 6G). When Matrigel plugs were harvested and vessel density documented, shRNA-224-expressing PMVECs displayed a high vessel density (Fig. 6, H and I). Thus, we were unable to reduce NAP1 expression in PMVECs for the time needed to complete the in vivo Matrigel experiments, although the rebound increase in NAP1 expression is consistent with evidence that increased NAP1 expression results in a proliferative and vasculogenic endothelial cell phenotype.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Lung microvascular endothelium is enriched with progenitor cells that underlie its rapid proliferation and increased vasculogenic capacity (4). This proliferative behavior represents a stable cell trait, as it is present in PMVECs studied in culture over many passages. The molecular basis for such an enhanced growth potential is unknown. Presently, we sought to determine whether NAP1 contributes to the growth and vasculogenic potential of endothelium. Consistent with this idea, we found that rapidly growing cells (PMVECs and RMEPCs) possess a high NAP1 abundance, and slow-growing cells (PAECs) possess a low NAP1 abundance, in both cytosolic and nuclear fractions. Heterologous NAP1 expression in slow-growing cells (PAECs) increases their growth potential, whereas inhibition of NAP1 expression in fast-growing cells (PMVECs) decreases their growth potential. Moreover, high NAP1 expression confers increased vasculogenic potential to endothelium. These findings directly incriminate NAP1 in control of the proliferative, vasculogenic endothelial cell phenotype.

NAP1 is a highly conserved protein (from yeast to eukaryotes), particularly in eukaryotes (for review see Ref. 32). Approximately 25% of the protein comprises acidic residues that are found within four discrete regions dispersed across the protein sequence. NAP1 possesses nuclear import and export sequences, consistent with its chaperone function. The functional protein exists as a homodimer and forms a unique ellipsoidal shape. NAP1 plays an important cell type-specific role in maintaining epigenetic inheritance, and, in particular, a proproliferative trait. Studies across a variety of species (yeast, Xenopus, and A. thaliana) indicate that the NAP1 family of proteins regulates cell proliferation (3, 7, 9, 18–20, 24, 26, 29, 31). NAP1 deletion changes expression of ~10% of Saccharomyces cerevisiae genes (increased or decreased by a minimum of 2-fold); the principally affected genes are located in a gene cluster (19). The simultaneous knockdown of two NAP1-related proteins (NRP1 and NRP2) in Arabidopsis changes expression of ~100 genes, many relating to either proliferation or apoptosis (31). These collective studies suggest that NAP1 within the nucleus influences a gene cluster(s) that is necessary to establish cell proliferative capacity, but does not change global cell phenotype. Our findings support this notion, as NAP1 abundance was an important determinant of cell growth, but did not change the expression pattern of endothelial cell markers, and did not change global endothelial cell function, such as the ability to form a semipermeable barrier. More specifically, NAP1 expression was not sufficient to change the PMVEC and PAEC imprint. Although NAP1 was originally described for its ability to remodel chromatin (13, 14), it has more recently been shown to chaperone the nuclear import of H2A and H2B (30), interact with p300 and regulate gene transcription (5, 15, 25), facilitate the exchange of histone variants in nucleosomes (30), and promote nucleosome sliding along DNA (21). Any or all of these functions may impact on how NAP1 determines which proliferative genes are actively transcribed, yet the organization of a proliferative gene cluster that is controlled by NAP1 and is inherited through mitotic cell divisions has not been specifically resolved, particularly in endothelium.

NAP1 is located within the cytosol and nucleus of interphase cells and within the nucleus of dividing cells. While NAP1 within the nucleus influences expression of proproliferative genes, NAP1 within the cytosol interacts with cyclins (3). Cyclin B activity is present during the G2/M transition and is necessary for successful formation of the mitotic spindle. NAP1 interacts with cyclin B and Gin4 in budding yeast, and both of these proteins are required for successful growth (3). While our studies resolve that NAP1 is an important determinant of the endothelial cell growth potential, we have not determined the extent to which this phenotype depends on transcriptional regulation of proliferative genes, such as VEGF, or interaction of NAP1 with cyclin B. It is noteworthy that microarray data and measurements of secreted protein indicate that PMVECs express and secrete considerably more VEGF than do PAECs, and NAP1 is present with p300 at the VEGF hypoxic responsive element in DNA affinity precipitation analysis (data not shown). It will be important to examine whether NAP1 interacts with p300 and H2A and H2B to facilitate the expression of VEGF, and other growth factors, to promote PMVEC proliferation.

We observed that NAP1 overexpression was a determinant of endothelial vasculogenic capacity. It is most likely that the proliferative behavior of NAP1 overexpressing cells is essential to this function, although we did not rule out the possibility that NAP1 contributes to other cellular processes equally important to new blood vessel formation, including matrix breakdown, migration, and vessel maturation. Our studies incriminate NAP1 in renewing aged and injured endothelium, but also bring into question whether NAP1 is important in developmental angio-/vasculogenesis and whether NAP1 dysfunction contributes to abnormal endothelial cell growth, as in hemangioma, hemangioendothelioma, angiosarcoma, telangiectasia, Kaposi's sarcoma, and plexiform lesions.

In summary, we have identified NAP1 as an important regulatory stimulus in endothelial growth and vasculogenic potential. Fast-growing cells express high NAP1, whereas slow-growing cells express low NAP1. NAP1 overexpression increases endothelial cell growth potential, and NAP1 inhibition decreases endothelial cell growth potential, without changing the global cell phenotype. Thus, our findings incriminate NAP1 expression and function in normal endothelial cell turnover and repair and suggest that NAP1 dysfunction could contribute to inappropriate endothelial cell growth responses.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-66299 and HL-60024 and Riley Children's Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Raymond Hester, Linn Ayers, Anna Buford, and Freda McDonald for contributions to the development of this work. We thank Drs. Ivan McMurtry, Mark Gillespie, and Brian Fouty for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Troy Stevens, Dept. of Molecular and Cellular Pharmacology and Center for Lung Biology, Univ. of South Alabama College of Medicine, Mobile, AL 36688 (e-mail: tstevens{at}jaguar1.usouthal.edu)

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.


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