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Involvement of Low‐Density Lipoprotein Receptor in the Pathogenesis of Pulmonary Hypertension

Background Recently, we and others have reported a causal role for oxidized lipids in the pathogenesis of pulmonary hypertension (PH). However, the role of low‐density lipoprotein receptor (LDL‐R) in PH is not known. Methods and Results We examined the role of LDL‐R in the development of PH and determined the efficacy of high‐density lipoprotein mimetic peptide 4F in mitigating PH. Explanted human lungs and plasma from patients with PH and control subjects were analyzed for gene expression, histological characteristics, and lipoprotein oxidation. Male LDL‐R null (LDL‐R knockout) mice (12–15 months old) were fed chow, Western diet (WD), WD with 4F, and WD with scramble peptide for 12 weeks. Serial echocardiography, cardiac catheterization, oxidized LDL assay, real‐time quantitative reverse transcription–polymerase chain reaction, and histological analysis were performed. The effect of LDL‐R knockdown and oxidized LDL on human pulmonary artery smooth muscle cell proliferation was assessed in vitro. LDL‐R and CD36 expression levels were significantly downregulated in the lungs of patients with PH. Patients with PH also had increased lung lipid deposits, oxidized LDL, E06 immunoreactivity, and plasma oxidized LDL/LDL ratio. LDL‐R knockout mice on WD developed PH, right ventricular hypertrophy, right ventricular dysfunction, pulmonary vascular remodeling, fibrosis, and lipid deposition in lungs, aortic atherosclerosis, and left ventricular dysfunction, which were prevented by 4F. Interestingly, PH in WD group preceded left ventricular dysfunction. Oxidized LDL or LDL‐R knockdown significantly increased proliferation of human pulmonary artery smooth muscle cells in vitro. Conclusions Human PH is associated with decreased LDL‐R in lungs and increased oxidized LDL in lungs and plasma. WD‐fed LDL‐R knockout mice develop PH and right ventricular dysfunction, implicating a role for LDL‐R and oxidized lipids in PH.


Real time PCR
Total RNA was purified from human lungs using the Trizol method. Two micrograms of total RNA were reverse transcribed to cDNA using the Omniscript RT kit (Qiagen), according to the manufacturer's instructions (Qiagen). Quantitative real-time reverse transcriptase-polymerase chain reaction (QRT-PCR) was performed using iTaq universal SYBR green supermix (Bio-Rad cat#1725121). The LDL-R and CD36 gene expression was assessed using gene-specific primers. GAPDH was used as a reference control for normalization. Levels of gene expression in each sample were determined with the comparative Ct method using Bio-Rad CFX manager 2.1 software.
The primer sequences were as follows: For gene expression, thermocycler protocol was as follows: polymerase activation: 30 s at 95°C, then denaturation 5s at 95°C, and annealing/elongation 90s at 60°C for 40 cycles.

Animals and treatments
Middle aged male LDL-R KO mice (12-15 months old) were fed either chow (n=16) or Western diet (WD, n=27) for 12 weeks. Mice on WD were randomly divided into three groups, WD (n=9), WD together with Apolipoprotein A1 mimetic (HDL mimetic) peptide D-4F (WF+4F group, n=11), or WD together with scramble peptide (WD+SCRM group, n=7) in drinking water for 12 weeks. A group of chow fed mice received 4F (chow+4F group, n=8, Fig. S1). Serial echocardiography was performed to monitor cardiopulmonary hemodynamics and the development of PH and RV dysfunction. Direct RV and LV catheterization was performed terminally and RV hypertrophy index was calculated as weight ratio of RV/(LV+IVS). Aorta, RV, LV and lung tissue were collected.

Echocardiography and cardiopulmonary hemodynamic measurements
Transthoracic echocardiography was performed to monitor cardiopulmonary hemodynamics using a Vevo 2100 high-resolution image system (VisualSonics, Toronto, Canada). Echocardiograms including B-mode, M-mode and pulsed wave Doppler images were obtained under isoflurane aesthesia. Ejection fraction (EF, %) was measured using M-mode echocardiographic images. A 30-MHz linear transducer was used to perform the pulmonary pulsed-wave Doppler echocardiography of PA flow. The probe was placed in a parasternal long-axis position to visualize the PA outflow tract. Pulsed flow Doppler imaging was then overlaid to observe the dynamics of blood flow through the PA valve.
PA acceleration time (PAAT) was determined by calculating time taken from the start of flow to maximal velocity using echocardiogram software (Vevo 2100 version: 1.5.0).
The right ventricular systolic pressure (RVSP) and left ventricular systolic pressure (LVSP) were measured directly by inserting a catheter  ADInstruments) connected to a pressure transducer (Power Lab, ADInstruments) into the RV or LV just before sacrifice. Briefly, for cardiac catheterization, the mice were anesthetized with isoflurane. The animals were placed on a controlled warming pad to keep the body temperature constant at 37 °C. After a tracheotomy was performed, a cannula was inserted, and the animals were mechanically ventilated. After a midsternal thoracotomy, mice were placed under a stereomicroscope (Zeiss, Hamburg, Germany) and a pressure-conductance catheter (model 1.4 F Millar SPR-671) was introduced via the apex into the RV or LV and positioned towards the pulmonary or aortic valve respectively. The catheter was connected to a signal processor (ADInstruments) and pressures were recorded digitally. After recording the pressures, heart and lung tissues were removed rapidly under deep anesthesia for preservation of protein and RNA integrity.

Gross histologic analysis, tissue preparation and imaging
The right ventricular (RV) wall, the left ventricular (LV) wall, and the interventricular septum (IVS) were dissected. RV, LV, IVS and lungs were weighed. The ratio of the RV to LV plus septal weight [RV/(LV + IVS)] was calculated as the Fulton index of RV hypertrophy. Lungs were fixed in 4% paraformaldehyde (PFA) in 0.1 M Na2HPO4 and 23 mM NaHPO4 (pH 7.4) for 4 h on ice. The tissue was then immersed in ice-cold 20% sucrose in 0.1 M Na2HPO4 and 23 mM NaHPO4 (pH 7.4) overnight to cryoprotect the tissue, mounted using OCT, and transversal 4-6μm sections were obtained with a cryostat. Paraffin embedded control lung sections, obtained from the UCLA pathology lab, were sectioned at 5µm. Images were acquired using a confocal microscope (Nikon).

Immunofluorescence and immunohistochemistry staining
Lung sections (5μm) were fixed in 4% paraformaldehyde. The sections were then washed with PBS+0.1% Tween20 three times and incubated with 5% normal goat serum in PBS for one hour to block the background. Following blocking, the sections were incubated with primary antibodies in PBS+2% normal goat serum at 4°C overnight. Sections were then washed with PBS three times, incubated with the secondary antibody in PBS+2% normal goat serum at room temperature for one hour. After washing the secondary antibody with PBS three times, the sections were mounted for imaging using Fluoromount-G with DAPI (Molecular Probes) (ThermoFisher cat#00-4959-52).
For immunohistochemistry, endogenous peroxidase activity was inhibited by incubating the lung sections with 0.3% H2O2 in PBS for 20 min at room temperature followed by washing with PBS thrice. The lung sections were then incubated with 10% normal goat serum in PBS containing 0.1% Triton for 30 min at room temperature to block the nonspecific binding. The sections were incubated with the appropriate primary antibodies in PBS+0.1% Triton+ 1% normal goat serum at 4 o C overnight, and washed 3 times with PBS+0.1% Triton. The sections were incubated with HRP-conjugated secondary antibody (1:200 dilution) in PBS+0.1% Triton+ 1% normal goat serum, for 1 h at room temperature, washed with PBS+0.1% Triton three times, and stained with 3,3'diaminobenzidine (DAB) as a substrate (10X DAB solution + stable buffer) for 5-10 min.
The DAB was rinsed with dH2O and stained with hematoxylin for 1 min, washed under running tap water for 10 min. Dehydration was then performed by incubating in 50%, 70%, 96%, 100% ethanol and xylene. Sections were mounted using Permount (Fisher Scientific). The images were acquired using a laser scanning confocal microscope (Nikon Eclipse E 400).

Masson's Trichrome and Oil-Red-O staining
Masson Trichrome staining was performed according to the manufacturer's protocol (Sigma), and images were acquired with a confocal microscope (Nikon).
For Oil-Red-O staining, frozen sections cut at 4-6m, were fixed in formalin, and briefly washed with running tap water for 1-10 mins. Sections were rinsed with 60% isopropanol and stained with freshly prepared Oil-Red-O working solution for 15 mins. Sections were rinsed with 60% isopropanol. Nuclei were lightly stained with 5 dips of haematoxylin.
Sections were rinsed with distilled water and mounted in aqueous mounting medium.
Images were acquired with a confocal microscope (Nikon).

Quantification of pulmonary vascular remodeling, lung fibrosis and lipid deposition
For assessment of pulmonary arteriolar wall thickness, only distal pulmonary arteries less than 100μm were quantified (3-4 vessels per mouse; number of mice: chow=7, WD=10, WD+Scramble=4, WD+4F=7) using ImageJ software. Stitched images of entire lung sections were obtained using 10x objective using a confocal microscope. Pulmonary arteriolar wall thickness was calculated by subtracting diameter of the lumen from total diameter of the vessel, divided by total diameter of the vessel. As the diameter of the vessel and lumen are not usually similar in different directions, pulmonary arteriolar wall thickness was measured in two different directions and averaged. Assessment of percent fully muscular, partially muscular and non-muscular arteries was also made (Fig. 5).
Pulmonary fibrosis was quantified using a grid over stitched images of lungs that divided the field of view into 100 squares, each collagenous tissue (blue stain) filled square in the grid was scored as 1 (present) or 0 (absent). Results are expressed as the percentage occupied by fibrosis to the total area examined (number of mice: chow=7, WD=11, WD+Scramble=7, WD+4F=6). Percent lipid deposition was quantified in lung sections using ImageJ software (number of mice: chow=4, WD=4, WD+Scramble=4, WD+4F=4).

Statistics
Unpaired T-tests and one-way ANOVA tests were used to compare between groups using GraphPad Prism for Windows. When significant differences were detected, individual mean values were compared by post-hoc tests (Tukey or Dunnett) that allowed for multiple comparisons. P < 0.05 was considered statistically significant. Values are expressed as mean ± SEM.