Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice

Significance Pulmonary arterial hypertension (PAH) is a disease in which lung blood pressure is raised chronically, causing right heart failure. It has been shown that iron deficiency also raises lung blood pressure. However, we don’t know the mechanisms because we don’t understand precisely how cells of the lung blood vessels are affected by iron levels. The smooth muscle cells of the lung blood vessels are important for controlling lung blood pressure. Our study shows that iron deficiency specifically within these cells is sufficient to cause PAH, even against a background of normal iron levels in other tissues.

Table S1-Parameters of LV function are not different between mice of the two genotypes.

Mice
Female fpnC326Y fl mice were mated with SMMHC-CreER T2 males, which are transgenic for Y chromosome-linked CreER T2 gene under control of smooth muscle myosin heavy chain polypeptide 11 (myh11) promoter (1). After 2 rounds of breeding, male fpnC326Y fl/fl SMMHC-CreER T2 + mice were identified and mated with female fpnC326Y fl/fl mice to generate fpnC326Y fl/fl SMMHC-CreER T2 + males used in experiments. Age-matched fpnC326Y fl/fl control males were generated from breeding fpnC326Y fl/fl males and females. Experimental fpnC326Y fl/fl SMMHC-CreER T2 + males and age-matched fpnC326Y fl/fl control males were induced between 3 and 4 weeks of age, using a total of 3 intraperitoneal injections delivering 1mg tamoxifen every second day.Unless otherwise stated, mice were fed a standard chow diet containing 200ppm iron. In systemic iron deficiency studies, animals were fed an irondeficient diet (5 ppm iron; Teklad TD.99397; Harlan Laboratories), or a matched control diet (200 ppm iron; Teklad TD.08713) from weaning for six weeks.

Acute hypoxia human study
Each study day entailed a 6-hour eucapnic hypoxic exposure (oxygen end-tidal partial pressure 55 mmHg) in a normobaric chamber. On the first study day, immediately before commencement of the hypoxic exposure, 0.9% saline was administered i.v., and on the second, 15 mg/kg (maximum 1 g) ferric carboxymaltose (Ferinject, Vifor Pharma) was added to an appropriate volume of 0.9% saline; each infusion was of 250 ml total volume and given over 15 minutes at a rate of 16.7 ml/min. Venous blood was sampled before each infusion and at 6 hours. Plasma was obtained by centrifugation and immediately frozen at -80°C.

PASMCs with BMPR2 mutations
Cells were a kind gift from Prof N Morrell, University of Cambridge. Mouse PASMCs were obtained from mice with a ubiquitous knock-in of R899X mutation (2). Human PASMCs were harvested from explanted lung tissue obtained from healthy controls and PAH patients with BMPR2 mutations: R899X, W9X and W930S. Patient demographics are detailed in Table  S2.

Isolation of mouse PASMCs
Following isolation in DM, mouse pulmonary arteries were digested by addition of 30 mg/ml papain (product code P4762, Sigma-Aldrich) for 1 h at 4°C, followed by 6 min incubation at 37°C in the presence of 1 mg/ml dithiothreitol (product code D0632, Sigma-Aldrich), then a further incubation with 20 mg/ml Collagenase (C2674, Sigma-Aldrich) for 5 min at 37°C. Tissues were washed in ice cold DM and gently triturated to release cells from the tissue matrix.

Gene expression
Gene expression was measured by quantitative real-time PCR, using Applied Biosystems Taqman gene expression assay probes for dmt1, tfr1 and et-1 and house-keeping gene β-Actin (Life Technologies, Carlsbad, CA). The CT value for the gene of interest was first normalised by deducting CT value for β-Actin to obtain a delta CT value. Delta CT values of test samples were further normalised to the average of the delta CT values for control samples to obtain delta delta CT values. Relative gene expression levels were then calculated as 2 -delta deltaCT .

Quantitation of vessel muscularization
One hundred pulmonary arterioles per section were analysed. Each vessel was assigned either as non-muscularized (no α-SMA staining), partially muscularized (thin or discontinuous layer of α-SMA staining), or fully muscularized (thick continuous layer of α-SMA staining). The percentage distribution of each was calculated per group.

Determination of RV and cardiomyocyte hypertrophy
Hearts were isolated from PBS-perfused fpnC326Y fl/fl SMMHC-CreER T2 + males and agematched fpnC326Y fl/fl control males at 6 and 12 weeks post-tamoxifen injection. Hearts were weighed on a precision balance and weights recorded before and after removal of the RV. Values were expressed as RV/LV+septum. For determination of cardiomyocyte size, FFPE heart sections were stained for wheat germ agglutinin WGA (W32464, Thermosfisher) at 5ug/ml, and counterstained with DAPI. Images of WGA-stained RVs were analysed by Image J to calculate the average size of RV cardiomyocytes for each mouse.

Iron indices
Serum iron and ferritin levels were determined using the ABX-Pentra system (Horiba Medical, CA). Determination of total elemental iron in tissues from PBS-perfused animals was carried out by inductively coupled plasma mass spectrometry (ICP-MS) as described previously (3,4). Haemoglobin was recorded from fresh blood using the HemoCue Hb 201+ system. Ferritin levels in cell lysates were measured by ELISA (orb408073, Biorbyt for mouse ferritin and LS-F5312, lsbio for human ferritin) according to the manufacturer's instructions.