Evidence supporting a role for circulating macrophages in the regression of vascular remodeling following sub-chronic exposure to hemoglobin plus hypoxia

Animals

Male Sprague-Dawley rats were obtained from a commercial vendor (Jackson Laboratories, Bar Harbor, ME, USA). All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at our University. All animals survived the surgical procedure and indwelling catheterization into the jugular vein. None of the rats exhibited any signs or symptoms indicative of systemic infection. After surgery, wounds healed within 14 days.

Rats (n = 28) were randomly assigned to one of four groups: (1) normoxic control (NX; n = 6); (2) hypoxic control (HX; n = 6); (3) hemoglobin plus hypoxia (Hb + HX; n = 8); (4) hemoglobin plus hypoxia treated with gadolinium chloride (Hb + HX + GdCl₃; n = 8).

iPrecio infusion pump placement

Programmable infusion pumps (iPRECIO, Tokyo, Japan) were placed subcutaneously as previously described.⁵ iPRECIO pumps were programmed to deliver Hb (35mg/day or 250 ug/uL) or saline at an infusion rate of 6 mL/h for 35 days. Pumps were refilled with fresh aliquots of Hb or saline every three to four days. At a dose of 35 mg/day, we show that total plasma heme concentrations were in the range of values similar to a mild to moderate chronic hemolytic state observed in SCD.^{5, 6}

Endotoxin and catalase-free human adult hemoglobin (HbA; (Fe²⁺))

Purified human endotoxin-free Hb (LPS < 0.5 endotoxin units, EU) was prepared from outdated blood as previously described, with an additional step to remove catalase by chromatography.⁹ Approximately 1 g of Hb protein was loaded onto a GE Healthcare glass column (HR 16/50:1 ½″ × 26″) packed with SuperoseÔ 6 (Code No. 17-0489-01, Amersham Biosciences, Upsala, Sweden) and run at 4°C and a flow rate of 2 mL/h. Hb protein was collected and was followed by buffer switching to 0.1 NaCl to remove catalase from the column. Several runs were pooled and concentrated to 200 mg/mL using Centricon Plus–70 with 30 kDa cutoff membrane filters (Millipore, Billerica, MA, USA). The starting composition of Hb was 96.5 ± 1.3% Fe²⁺, 3.50 ± 0.23% Fe³⁺, and no measurable hemichrome. Endotoxin concentration was <0.5 EU/mL as determined by the limulus ambocyte lysate assay and failed to activate macrophages in culture.

Hypoxia exposure

Rats were continuously exposed to simulated high altitude (5500 m, 18,000 ft) in a rodent hypobaric chamber facility as previously described.^5-7 Animals were exposed to this atmosphere continuously for 35 days. It has been previously demonstrated that chronic hypoxia can increase pulmonary arterial pressure, right ventricular hypertrophy, and pulmonary vascular remodeling.

Echocardiography

To assess approximate time frames of the progression and resolution of PH echocardiographic analysis was performed at two-week intervals using a high-resolution in vivo microimaging Vevo770 system (Visual-Sonics). Care was taken to follow the guidelines for measuring cardiac physiology in rats established by the American Physiological Society.¹⁰ For each analysis, rats were anesthetized via an isoflurane/O₂ mixture with induction at 4% and maintenance between 1.5% and 3%, and body temperature was maintained at 37°C via a heated surgical pad. As described by Cavasin et al.,¹¹ pulse-wave Doppler imaging of the pulmonary outflow was recorded in the parasternal short-axis view at the level of the aortic valve. Animals were then monitored during the recovery from anesthesia; upon full recovery, they were returned to their respective housing

Gadolinium chloride

Gadolinium chloride (GdCl₃) (Cat # G7532; Sigma) was dissolved in sterile 0.9% NaCl and delivered intravenously in a 1 mL bolus. Rats were treated with gadolinium chloride every three days at a dose of 10 mg/kg for four weeks. We previously demonstrated by qRTPCR analysis that this dose effectively depletes macrophages by depleting CD163⁺ and CD68⁺ cells in the liver.⁷

Endpoint blood pressure measurements

Pulmonary blood pressures were measured with a 1.9F pressure-volume catheter placed in the main PA in an open-chest procedure. Correct placement of the catheter was confirmed by observing the normal rise in diastolic pressure as the catheter was moved into the PA. Systemic blood pressure was monitored with a catheter inserted in the femoral artery. Cardiac performance was assessed using an ADV500 pressure-volume system (Transonic/Scisense), and data acquired and analyzed with Labscribe2 (iWorx). For all measurements, animals were anesthetized with 5% and maintained at 2% isoflurane, and body temperature was maintained at 37°C.

Blood and organ collection

Serial blood draws were conducted at baseline (pump placement) and every three days post initiation of Hb and hypoxia exposure. Blood was placed in an EDTA-K + vacutainer and a crit tube to analyze plasma and hematocrit, respectively.

Animals were exsanguinated via a carotid artery catheter; blood was placed in a vacutainer with EDTA and centrifuged. The plasma was collected, snap-frozen in liquid nitrogen, and stored at –80°C until analysis; 300 μL of blood was also placed in a crit-tube and spun to measure hematocrits. The femoral artery was severed, and the lungs, kidneys, spleen, and liver were perfused with PBS (120mL) via the right ventricle to remove blood. The kidneys, spleen, and liver were cut in two sections: one piece was placed in 10% formalin and the other in AllProtect (Qiagen, Valencia, CA, USA). The right lung lobes were tied off at the right main bronchus, removed, and a piece was placed in AllProtect, and another portion was snap-frozen. The left lung was fixed with 10% formalin (∼3 mL) by airway inflation under constant pressure at 25 cm H₂O pressure, and the entire lung-heart bloc was removed. The hearts were removed, and the right and left ventricles plus septum were isolated. The right ventricle (RV) and left ventricle with the septum (LV + S) were weighed for assessment of the Fulton Index (right ventricular hypertrophy; RV/LV + S). After 24 h, the formalin-fixed organs were removed from 10% formalin and placed in 70% ethanol, and prepared following standard methods for morphometric and immunohistochemical analyses.

Materials

The following antibodies were used: b actin (cat # ab8226), 1:1000 (Abcam); smooth muscle actin (cat #ab5694 and ab7817), 1:1000 (Abcam); heme oxygenase (HO1; cat # ab13248), 1:250 (Abcam); interleukin 6 (IL6; cat # ab9324), 1:1000 (Abcam); endothelin 1 (ET1; cat # ab117757), 1:500 (Abcam); CD68 (cat # ab955), 1:500 (Abcam); intracellular adhesion molecule 1 (ICAM; cat # ab2213), 1:250 (Abcam). Lung sections were incubated with a fluorescent secondary antibody either AlexaFluor 488; 1:1000 or AlexaFluor 555; 1:1000 (Invitrogen; Carslbad, CA, USA) or a biotinylated secondary horse anti mouse or horse anti-rabbit, 1:1000 (Vector Labs, Burlingame, CA, USA).

Western blot analysis

Lungs were homogenized using a TissueMiser (ThermoFisher, Waltham, MA, USA) in RIPA buffer containing protease inhibitors; protein concentrations were determined using a Pierce BCA Protein Assay. Briefly, Western blot analysis was performed using 30 mg of sample protein run under denaturing and reducing conditions on Biorad Criterion Tris-HCl 12.5% gels with a Biorad SDS-Page blot system. All proteins were normalized to b-actin. Statistical analysis was determined from the fold difference from Nx (normoxic control) on the same gel. Gels were imaged on an Alpha Innotech gel documentation system (Protein Simple, Santa Clara, CA, USA), and densitometric analyses were performed using ImageJ software (version 1.44o, National Institutes of Health, USA).

Immunohistochemistry and morphology

Formalin-fixed, paraffin-embedded lung, kidney, and spleen tissue was sectioned by our university. Five-micrometer sections were stained with hematoxylin and eosin by standard procedures to assess the accumulation of perivascular cells as well as vessel wall thickness. Lung sections were also stained for smooth muscle actin utilizing a biotinylated horse anti-mouse immunoglobulin followed by a 3,3′-diaminobenzidine (DAB) for development to quantify small arterial muscularization.

The entire tissue field on each slide was scanned utilizing a Leica AperioCS2 microscope. Analyses of the scanned images were performed using the Aperio eSlide manager software (Leica, Buffalo Grove, IL, USA). Positively stained smooth muscle vessels were counted and described as either fully or partially muscularized. Any area that fell outside of the lung boundary was subtracted from the total area, and the number of either fully or partially muscularized vessels was normalized to area. Distal pulmonary vessels (outside diameter of 10–50 mm) were assessed for degree of circumferential a-smooth muscle actin-positive staining, indicative of muscularization. Proximal vessels (outside diameter of 50–250 mm) were analyzed for medial wall thickness at four points around the vessel circumference and for lumen diameter along two axes. Wall thickness is expressed as the ratio of medial wall thickness to lumen radius.

Histological analysis of nonheme iron deposition in lung tissue was detected using Perl's staining with DAB counterstain as previously described.^5-7 For quantification, a total of 20 images at 20× magnification were obtained from sections of all animals. Within each of the 20 images, the total number of Perl's positive cells was counted and divided by 20.

Statistical analysis

For all groups, the mean SEM is reported. Statistical comparisons for data measurements were completed with either one-way analysis of variance¹² for NX, HX, Hb + HX, and Hb + HX + GdCl₃ treatment (endpoint analysis) or two-way ANOVA for analysis between time (echocardiography analysis). Echocardiography data were also analyzed by a univariate repeated measures test using JMP (SAS Cary, NC, USA). All Post hoc analyses were completed with unpaired, two-sided Student t-test with a Bonferroni correction unless otherwise noted. Statistical analyses were performed using GraphPad Prism (La Jolla, CA, USA) with statistical significance set at p ≤ 0.05.

Macrophage depletion slows the resolution of pulmonary vascular remodeling in a hemoglobin plus hypoxia rat model of pulmonary hypertension

Hypoxia-induced vascular remodeling reverses upon reoxygenation. We demonstrate that rats exposed to HX with continuous low-level Hb infusion for five weeks develop a more severe PH than with Hx alone.^5-7 In this study, we wanted to assess whether our PH model would be reversible after the withdrawal of Hb and HX and if macrophages have a role in this process. To this end, circulating monocytes/macrophages were depleted with GdCl₃ during a four-week recovery period after cessation of both Hb and HX (Fig. 1a). Groups consisted of normoxic control (NX), hypoxic control (HX5wk), (Hb + HX5wk), and HX 5wk followed by four weeks normoxia (HX5wk; NX+4wk), Hb + HX 5wk followed by four weeks no Hb infusion in normoxia (Hb + HX5wk; -Hb + HX4wk) ± GdCl₃ (Fig. 1b). Serial echocardiography and endpoint hemodynamics, Fulton index (RV/LV + S), and lung morphology/histology were used to assess the impact of macrophage depletion on PH resolution. The purpose of echocardiography was to understand approximate time frames of the progression and resolution of PH, and not as a primary indicator to identify exact differences among groups more suitable for endpoint analysis.

Echocardiography analysis revealed that Hb + HX or HX exposed rats demonstrated increased pulmonary artery acceleration time (PAAT) and RV wall thickness compared to NX cohorts at five weeks. Combining all Hb + HX rats at five weeks before GdCl₃ treatment revealed lower PAAT in Hb plus HX rats than rats only exposed to Hx, reflecting higher PA pressures (Fig. 1b, inset) and more progressive PH (Fig. 1b–d). This main effect of Hb + HX is congruent with our previous studies demonstrating higher pulmonary arterial pressures in Hb plus HX rats vs. HX alone.^{5, 6} Analysis of serial echocardiography data shows a significant improvement of PAAT and RV wall thickness during the four-week recovery period (Fig. 1b–d). However, rats maintained on continuous Hb infusion plus HX had lower PAAT and greater RV wall thickness at two- and four-week time points of recovery (Fig. 1b and c) than HX alone rats (Fig. 1b–d).

Invasive hemodynamic measurements obtained after four weeks of recovery show the elimination of circulating macrophages with GdCl₃ prevented the complete resolution of mean pulmonary artery pressure (PAP) and pulmonary vascular remodeling as compared between the Hb + HX and Hb + HX + GdCl₃ groups (Fig. 1e–h). These data suggest that a subset of circulating monocytes/macrophages recruited to the lung may be required for the complete regression of Hb + HX-induced vascular remodeling. While the Fulton index calculation (RV/LV + S) demonstrated RV hypertrophy was not completely resolved in any group (Fig. 1g), the comparisons between Hb + HX and Hb + HX + GdCl₃ did not reach statistical significance. However, by comparing HX + GdCl₃ treated animals compared to HX alone, we noted greater mean PAP, medial thickening, and Fulton index (Supplemental Data Fig. 1). When combining all the treated and non-treated GdCl₃ animals into two groups, data suggest eliminating circulating macrophages with GdCl₃ slows the resolution of PAP, and medial thickening, RV hypertrophy, at four weeks (Supplemental Fig. 1). While other factors are at play in the reversal of PH, taken together these data suggest the circulating macrophage may work in concert with other cell types to either hasten the reversal process or are needed for complete resolution.

Gadolinium decreases cd163 positive but increases cd68 positive macrophages in the lung after four weeks of reoxygenation

We demonstrate iron accumulation in the lung adventitial macrophages by five weeks of chronic Hb exposure with concomitant HX, and depletion of circulating macrophages prevented PH development in this model.^5-7 Initially, we sought to determine whether macrophages containing iron persist at four weeks after cessation of the stimulus. Tissue Perls-DAB staining of the lung revealed that some iron accumulation persists in the adventitia and parenchymal cells in the Hb + HX rats (Fig. 2a). However, we cannot conclude if the early iron-containing macrophages survive in the lung for extended periods. As expected, we observed virtually no iron in the NX control animals and minimal iron in the HX and Hb + HX + GdCl₃ cohorts (Fig. 2a). Macrophage activation and polarization to M2 phenotype promote PH and vascular remodeling. We characterized the macrophages in the lungs by staining for cd68, a pan macrophage marker, and cd163, a marker for the M2 phenotype. Levels of cd68, a pan macrophage marker, were lower in HX and Hb + HX recovery groups but was significantly higher in Hb + HX + GdCl₃ treated recovery group (Fig. 2b and c). Staining for cd163, a marker for M2 macrophage shows a significant decrease in the Hb + HX and Hb + HX + GdCl₃ recovery group but was not affected in the HX group compared to the HX group five-week time point (Fig. 2d–e). These data demonstrate that during regression, there is a decrease in the M2 macrophage phenotype. Interestingly there is also an increase in a subset of macrophages that appear resistant to GdCl₃ treatment in the Hb + HX group (Fig. 2b–c).

Decreased IL-6 and ET1 expression in rat lungs during recovery

In rats exposed to Hb + HX for five weeks, there is increased iron accumulation in adventitial macrophages that express mediators of hypoxia-driven PH, IL-6, and ET-1.⁷ Here, we examined if IL-6 and ET-1 expression persisted in the residual iron overloaded macrophages after reoxygenation. Western blot analysis of lung lysates revealed decreased IL-6 in all groups of the recovery cohort. Furthermore, we observed IL-6 positive stain was predominantly in the media of distal pulmonary arteries of Hb + HX treated rats during regression and not in the iron-laden macrophages (Fig. 3a–c).

In contrast to IL-6, we observed robust ET-1⁺ cells in Hb + HX rats located in both the adventitial and vascular compartments in distal pulmonary arteries (Fig. 3d–f). These areas were generally in the same locations as we observed and Iron⁺ cells, suggesting ET-1 persistence may be associated with iron-loaded macrophages (Fig. 3f). ET-1 was decreased in the Hb + HX + GdCl₃ group, suggesting macrophages may be the dominant source of ET-1 in the lung (Fig. 3d). As expected, we observed minimal expression of Il-6⁺ and ET-1⁺ cells in both NX control rats and rats one month after chronic HX exposure. These results suggest that circulating macrophages increase ET-1 levels, but IL-6 levels were driven by hypoxia. Overall, these data demonstrate that the circulating macrophage influences PH mediators differentially during regression.

Increased HO-1 expression during recovery

Expression of HO-1 is considered protective in PH and other vascular diseases. We observed the persistence of HO-1 in iron positive cells in the vessel wall, in the Hb + HX group like the macrophage expressing the Hb clearance phenotype in our previous studies.^5-7 Interestingly, eliminating circulating macrophages during regression resulted in increased expression of HO-1⁺ in cells throughout the lung (Fig. 4a–c). HO-1⁺ cells are distributed throughout the vessel wall and highly expressed in the media of distal pulmonary arteries of the Hb + HX + GdCl₃ rats (Fig. 4a–c), demonstrating a shift in lung HO-1 cellular expression with depletion of circulating macrophages. Congruent with immunofluorescence staining, Western blot analysis of whole lung tissue confirmed that HO-1 expression was significantly greater in the Hb + HX + GdCl₃ treated rats (Fig. 4c). These results differ from those observed in the preventive study with GdCl3 treatment, where HO-1 expression did not increase in the vessel wall. In the NX and HX cohorts, we noted HO-1 expression was low in parenchymal and adventitial cells of the lung (Fig. 4a–c). Within the five-week main study, Hb + HX caused diffuse fibrosis in peripheral pulmonary vessels consistent with muscularized remodeled lung vasculature (Fig. 4d). Fibrosis was not nearly as extensive in NX or the HX groups. During the four-week regression phase, Hb + HX + GdCl₃ treated rats demonstrated diffuse interstitial and muscularized tissue fibrosis (Fig. 4d), suggesting that depletion of peripheral macrophages was not a factor in the progression of fibrosis during the four-week regression phase.