Mast cell number, phenotype, and function in human pulmonary arterial hypertension

Abstract

A proliferation of mast cells around the small pulmonary blood vessels and the alveolar septae has been noted in models of pulmonary hypertension, and in plexiform lesions of pulmonary arterial hypertension (PAH) in patients. Here, we hypothesize that total mast cell numbers and activation are increased in PAH and that they contribute to vascular remodeling through cellular and soluble proangiogenic effectors. To test this, blood and urine were collected from patients with PAH (N=44), asthma (N=18) and healthy controls (N=29) to quantitate biomarkers of total body mast cell numbers and activation (total and mature tryptase, N-methyl histamine, leukotriene LTE(4) and prostaglandin PGD-M). Serum total tryptase was higher in PAH than that in controls suggesting greater numbers of mast cells, but indicators of mast cell activation (mature tryptase, LTE(4) and PGD-M) were similar among PAH, asthma, and controls. Immunohistochemistry of lung tissues identified mast cells as primarily perivascular and connective tissue chymase(+) type in PAH, rather than mucosal phenotype. Intervention with mast cell inhibitors cromolyn and fexofenadine was performed in 9 patients for 12 weeks to identify the influence of mast cell products on the pathologic proangiogenic environment. Treatment decreased total tryptase and LTE-4 levels over time of treatment. This occurred in parallel to a drop in vascular endothelial growth factor (VEGF) and circulating proangiogenic CD34+CD133+ progenitor cells, which suggests that mast cells may promote vascular remodeling and dysfunction. In support of this, levels of exhaled nitric oxide, a vasodilator that is generally low in PAH, increased at the end of the 12-week mast cell blockade and antihistamine. These results suggest that mast cells might contribute to the pulmonary vascular pathologic processes underlying PAH. More studies are needed to confirm their potential contribution to the disease.

Keywords: mast cells; proangiogenic progenitor cells; pulmonary arterial hypertension; tryptase.

Figure 1

Increased numbers of connective tissue type mast cells in PAH lungs. (A–H) Tryptase and chymase immunostaining of explanted control and PAH lungs. (A–E) Tryptase stained lung tissue sections. (F–H) Chymase stained lung tissue sections. Explanted PAH and failed donor (control) lung paraffin embedded tissue sections were immunostained for tryptase and chymase to identify and characterize mast cell phenotype (brown cells). Mast cell numbers were increased in the lungs of patients with PAH compared to controls (P=0.01) and localized predominantly to perivascular regions as opposed to submucosal regions as in control lungs. Mast cells in PAH lungs were tryptase+ and chymase+ consistent with a connective tissue phenotype as opposed to primarily tryptase+ in control lungs. Panels A–B – Control lungs stained for tryptase: (A) Mast cells are seen in the submucosal regions of the airways of control lungs. (B) Mast cells around a blood vessel in a control lung are less compared to PAH lungs. Panels C–H – PAH lungs: (C–E) Tryptase+ mast cells in PAH are in the perivascular adventitia and increased in number. (F–H) Mast cells in the perivascular regions are chymase+. In panels E–G where plexiform lesions are noted, mast cells are seen within the lesions. Magnification: (1) 5×, (2) 10×, (3) 20×, (4) 40×. Scale bar: 25μm.

Figure 2

(A) Serum total tryptase levels are higher in PAH than controls. Total serum tryptase levels are higher in PAH compared to controls indicating greater total numbers of mast cells in PAH. Box plots indicate median values, upper and lower quartiles. (B) Serum total tryptase levels correlate with brain natriuretic peptide (BNP). Total tryptase levels are related to disease severity assessed by BNP.

Figure 3

Mast cell blockade for 12 weeks with cromolyn and fexofenadine. Mast cell activation and proangiogenic biomarkers were measured at baseline Week 0 (prior to start of therapy). Four weeks later medications were started, and patients evaluated after 4, 8, and 12 weeks of therapy. Total tryptase and urinary LTE₄ dropped with mast cell blockade therapy. Proangiogenic CD34⁺CD133⁺ myeloid progenitors and VEGF also decreased with mast cell blockade. Asterisk significant changes compared to baseline (all P<0.05).

Figure 4

(A) Nitric oxide increases with mast cell blockade. Exhaled NO measured over the course of the study suggests improved pulmonary vascular health near the end of the 12-week therapy. The asterisk represents significant change from week 8 to week 12 (P<0.05). (B) NO levels are inversely correlated to VEGF. The inverse relationship suggests that the decrease of VEGF, which occurs with mast cell blockade, was associated with increasing levels of NO. Points are derived from all time points in the study.