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. 2019 Mar 18;14(3):e0213890.
doi: 10.1371/journal.pone.0213890. eCollection 2019.

Endothelial cells are a source of Nestin expression in Pulmonary Arterial Hypertension

Aneel R Bhagwani  1 , Schuyler Hultman  1 , Daniela Farkas  1 , Rebecca Moncayo  1 , Kaivalya Dandamudi  1 , Arsema K Zadu  1 , Carlyne D Cool  2 , Laszlo Farkas  1
Affiliations

Endothelial cells are a source of Nestin expression in Pulmonary Arterial Hypertension

Aneel R Bhagwani et al. PLoS One. .

Abstract

Uncontrolled proliferation of endothelial cells is essential to the pathogenesis of pulmonary arterial hypertension (PAH). Both proliferation and cytoskeleton reorganization are associated with upregulation of the intermediate filament protein Nestin. Recently, accumulation of Nestin-expressing cells was found in pulmonary vascular lesions of PAH patients. The goal of this study is to determine if Nestin expression contributes to endothelial proliferation in pulmonary arterial hypertension, using both lung tissues and endothelial cells. Here we found that endothelial cells from complex and plexiform lesions of PAH patients expressed Nestin. These Nestin+ cells further stained positive for the angiogenic factors CXC chemokine ligand 12 and Wnt1. Likewise, in the chronic hypoxia/SU5416 animal model of pulmonary hypertension, Nestin+ endothelial cells were found in occlusive pulmonary vascular lesions. In vitro, both growing rat and human lung endothelial cells expressed Nestin protein. When Nestin was overexpressed in endothelial cells (both rat and human), Nestin overexpression promoted proliferation and expression of CXC chemokine ligand 12. Nestin overexpression further increased angiogenic tube formation in vitro. Conclusions: We found increased Nestin expression from endothelial cells of occlusive lung vascular lesions in severe pulmonary hypertension. Elevated Nestin expression likely contributes to unchecked pulmonary vascular proliferation and angiogenesis, possibly via induction of CXC chemokine ligand 12. Additional studies are required to determine whether _targeting Nestin would be beneficial to treat PAH.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Endothelial cells are a source of Nestin expression in the remodeled pulmonary arteries from iPAH patients.
(A) Representative merged immunofluorescence (IF) images of optical sections and Z-stacks obtained by confocal microscopy show Nestin/α-SMA/vWF and Nestin/CD31. Note that many Nestin+ cells were also vWF+ (overlap of green and magenta pseudocolors, shown in white—upper row) or CD31+ (overlap of red and green pseudocolors, shown in yellow—lower row) in the pulmonary arteries from iPAH patients (arrows). Nestin staining was infrequent in control lung tissue (no pulmonary vascular disease). For the iPAH group, the left image shows an overview of the pulmonary artery/vascular lesion. The center image demonstrates the area indicated by a dotted box in more detail including orthogonal views of the z-Stack in XZ and YZ directions. The image on the right shows a projection of all z-stack images. The thin yellow and white lines indicate the location of reslicing on the X-, Y- and Z-axis. Arrows show representative Nestin+ vWF+ and Nestin+ CD31+ cells. Scale bar: 50 μm (overview images), 25 μm (detail images). Nuclear counterstaining with DAPI. Fluorochromes and pseudocolors: Nestin [AF647 (magenta), AF488 (green)], α-SMA [AF594 (red)], vWF [AF488 (green)], CD31 [AF647 (red)]. (B-C) Quantification of the fraction of Nestin+ cells (B) and Nestin+ vWF+ (C) in the pulmonary arteries/lung vascular lesions. N/M: non-muscularized/muscularized pulmonary arteries, C/P: pulmonary arteries with concentric or plexiform lesion. Note that there are two N/M groups: white N/M bars represent controls and blue N/M bars represent iPAH N/M vessels. Graphs in (B-C) demonstrate analysis using average for each patient in each group. Each bar represents the mean+SEM of the average fraction of positive cells in pulmonary vessels categorized according to type of vessel remodeling (N/M or C/P) per patient. N numbers used for statistics were the numbers of patients: controls: N/M n = 3, iPAH: N/M and C/P n = 6. *P<0.05 (Kruskal-Wallis). The total number of pulmonary arteries: control: N/M n = 30; iPAH: N/M n = 41; C/P n = 19.
Fig 2
Fig 2. Nestin+ cells express angiogenic factors in iPAH pulmonary artery lesions.
(A) Representative merged images of optical sections and z-stacks obtained by confocal microscopy show double IF stainings for Nestin/CXCL12 and Nestin/Wnt1. CXCL12 expression localized to pulmonary arterial lesions in iPAH lungs. It further co-localized (yellow) with Nestin in the vascular lesions (arrow). Control vessels had only scattered CXCL12 staining. Nestin+ Wnt1+ cells (yellow) were present in the pulmonary arterial lesions from iPAH patients (arrow). Nestin staining was absent in a control pulmonary artery. For the iPAH group, the left image is an overview of the pulmonary artery/vascular lesion. The center image demonstrates the area indicated by a dotted box in more detail. It also shows orthogonal views of the Z-Stack in XZ and YZ directions. The image on the right is a projection of all z-stack images. The thin white lines indicate the location of reslicing on the X-, Y- and Z-axis. Arrows show representative Nestin+ CXCL12+ and Nestin+ Wnt1+ cell. Scale bar: 50 μm (overview images), 25 μm (detail images). Nuclear counterstaining with DAPI. Fluorochromes and pseudocolors: Nestin [AF647 (red)], CXCL12 [AF488 (green)], Wnt1 [AF488 (green)]. (B-C) Quantification of the fraction of CXCL12+ Nestin+ cells (B) and Wnt1+ Nestin+ cells (C) in the pulmonary arteries/lung vascular lesions. N/M: non-muscularized/muscularized vessels, C/P: concentric or plexiform lesion. Note that there are two N/M groups: white N/M bars represent controls and blue N/M bars represent iPAH N/M vessels. Graphs in (B-C) demonstrate analysis using average for each patient in each group. Each bar represents the mean+SEM of the average fraction of positive cells in pulmonary vessels categorized according to type of vessel remodeling (N/M or C/P) and group (control vs. iPAH) per patient. N numbers used for statistics were the numbers of patients: controls: N/M n = 3, iPAH: N/M and C/P n = 6. *P<0.05 (Kruskal-Wallis). The total number of pulmonary arteries: control: N/M n = 30; iPAH: N/M n = 36 (CXCL12), n = 44 (Wnt1); C/P n = 22 (CXCL12); n = 16 (Wnt1).
Fig 3
Fig 3. Nestin expression in a rat model of severe PH.
(A) Representative merged double IF images of optical sections (naïve) and representative orthogonal views of Z-stacks (SU5416, cHx and cHx/Su) obtained by confocal microscopy show the localization of Nestin+ cells in pulmonary arteries. Staining further shows expression of endothelial markers vWF and VE-cadherin, or PASMC marker α-SMA. The image on the left shows a representative pulmonary artery of a naïve rat for each staining. On the right side, a projection of the complete Z-stack is shown for the cHx/Su 6 weeks images. Arrows point to representative Nestin+ vWF+, Nestin+ VE-cadherin+, and Nestin+ α-SMA+ cells. The thin white lines show the location of reslicing in X-, Y- and Z-direction. Scale bar: 20 μm (naïve), 25 μm. Nuclear counterstaining with DAPI. Fluorochromes: Nestin (AF488), vWF (AF594), VE-cadherin (AF594), α-SMA (AF594). (B) Quantitative RT-PCR of Nes mRNA expression in the lung tissue homogenate of naïve rats, rats exposed to cHx (3 weeks) and the cHx/Su protocol (3 and 6 weeks). (C) Quantitative analysis of the fraction of Nestin+ cells in pulmonary arteries using immunohistochemistry for Nestin in lung tissue sections from naive rats, rats exposed to cHx (3 weeks) and the cHx/Su protocol (3 and 6 weeks). (D) Right ventricular systolic pressure (RVSP) for the different groups confirm PH in cHx and cHx/Su rats. Each bar represents the mean+SEM of n = 3–4 animals. *P<0.05, **P<0.01 (Kruskal-Wallis).
Fig 4
Fig 4. Rat and human lung endothelial cells express Nestin.
(A) Representative flow cytometry of rat lung ECs for CD144 (Vascular Endothelial-cadherin) and VEGFR2. Rat lung ECs were negative for myeloid/hematopoietic markers CD133 and CD11b/c. The specific antibody staining is red, and the corresponding isotype is grey. (B) Rat lung ECs bind Griffonia simplicifolia lectin (G.s.), indicating microvascular ECs. (C) Rat lung ECs grown on chamber slides express Nestin. Note the perinuclear localization and the filaments extending throughout the cytoplasm. Control means omission of primary antibody. (B-C): Scale bars: 50 μm. (D) Representative Western blot showing Nestin expression in HLMVECs (β-actin as loading control). (E) Representative Western blots showing PCNA expression in HLMVECs (α-tubulin as loading control). Experiments 1–3 indicate unstimulated cells grown in separate dishes in EGM-2MV for Western blot analysis.
Fig 5
Fig 5. Nestin overexpression promotes endothelial proliferation.
(A) Co-immunofluorescence staining of PCNA and Nestin reveals multiple Nestin+ PCNA+ cells (arrow) in the intima of a concentric lesion from a PAH patient. In contrast, no Nestin+ PCNA+ cells were detected in the intima of a pulmonary artery from a control subject. Scale bars: 50 μm (overview), 25 μm (detail images). For PAH, the image on the left shows an overview of the blood vessel, whereas the images in the middle demonstrate orthogonal views of z-stacks from the area indicate by a dotted box. The image on the right is an intensity projection of the whole z-stack. (B) Transient Nestin overexpression in rat lung ECs 72h after adenoviral transduction (qRT-PCR). β2-microglobulin (B2m) was used as housekeeping gene. n = 6 per group. (C-D) Increased BrdU incorporation over 4 h following Nestin overexpression in rat lung ECs (72h after adenovirus inoculation). (C) Representative histograms and (D) quantification. n = 6 per group. (E) Transient Nestin overexpression (qRT-PCR) in human control PAECs 72h after adenovirus transduction (qRT-PCR). TATA-binding protein (TBP) was used as housekeeping gene. (n = 4 per group). (F) qRT-PCR of MKI67 (Ki-67, a proliferation marker) and CXCL12 in hPAECs 72h after AdNES or AdDL70 treatment. n = 4 per group. All data shown as mean+SEM. *P<0.05, **P<0.01 (Mann-Whitney). (G) Representative Western blots demonstrating the transgenic Nestin protein expression and elevated CXCL12 protein expression in PAECs 72h after adenoviral transfection with AdNES vs. AdDL70. β-actin was used as loading control.
Fig 6
Fig 6. Serum starvation reduces proliferation and induces apoptosis and Nestin expression in human PAECs.
(A) qRT-PCR expression of MKI67 (Ki67) in human PAECs cultured for 24h with complete EGM-2 or basal EGM (= serum starvation). (B-C) 24h serum starvation reduces proliferation in PAECs. (B) Representative histograms and (C) quantification. (D-E) 24h serum starvation promotes apoptosis measured as Annexin V (AV)+ 7-aminoactinomycin (7-AAD)- cells using flow cytometry. 7-AAD was added to exclude necrotic cells, which are 7-AAD+. (D) Representative dot plots and gating. (E) Quantification. (F) qRT-PCR of mRNA expression of NES and CXCL12 after 24h serum starvation. (G) Representative Western blots show that serum starvation promotes Nestin protein expression and increases caspase-3 cleavage, but not CXCL12 protein expression. (H) Quantification of Western blots in (G). n = 3 per group (H), n = 4 per group (E), n = 6 per group (A, C, F). All data shown as mean+SEM. *P<0.05, **P<0.01 (t-test, Mann-Whitney).
Fig 7
Fig 7. Nestin overexpression promotes angiogenesis in Matrigel.
PAECs were treated with AdDL70 or AdNES, or left untreated. After 48h, cells were seeded on Matrigel. (A) Representative images of PAECs after 19h on Matrigel in Ibidi μ Angiogenesis plates. Scale bar: 500 μm. Note that the images of whole wells were obtained using the manual stitching function in Olympus CellSens software. (B-E) Quantification of total tube length, number of nodes, number of branches and total branching length. Mean+SEM (n = 4–5 per group). *P<0.05, **P<0.01. Statistics were calculated using Mann-Whitney for AdDL70 vs. AdNES.
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