Vascular remodeling is airway generation-specific in a primate model of chronic asthma

Abstract

Rationale: Changes in the density of bronchial vessels have been proposed as a part of airway remodeling that occurs in chronic asthma.

Objectives: Using an established nonhuman primate model of chronic allergic asthma, we evaluated changes in vascular density as well as the contribution of bronchial epithelium to produce vascular endothelial growth factor (VEGF).

Methods: Eight juvenile rhesus macaques were divided into two groups of four. One group was exposed to 11 cycles of aerosolized house dust mite allergen (HDMA), whereas the other was exposed to filtered air. Bronchial wall vasculature was identified using an immunohistochemical approach, and vascular density was quantified stereologically. A semiquantitative polymerase chain reaction approach was used to estimate VEGF splice variant gene expression at discrete airway generations. Cell culture of primary tracheal epithelial cells with varying concentrations of HDMA was used to quantify the direct contribution of the epithelium to VEGF production.

Results: Bronchial vascular density was increased at mid- to lower airway generations, which was independent of changes in the interstitial compartment. The VEGF121 splice variant was significantly increased at lower airway generations. VEGF protein increased in a dose-dependant fashion in vitro primarily by an increase in VEGF121 gene expression.

Conclusion: This study highlights that increased vascular density in an animal model of chronic allergic asthma is airway generation specific and associated with a unique increase of VEGF splice variant gene expression. Airway epithelium is the likely source for increased VEGF.

Figure 1.

Protocol timeline. Schematic outline of sensitization and challenge protocol of juvenile rhesus monkeys to achieve state of allergic airway disease. The experimental exposure protocol we used is reflective of the episodic nature of allergen exposures with exposure to house dust mite allergen (HDMA) for 2 h on Days 3, 4, and 5 of each cycle. The exposure protocol for one 14-d cycle is illustrated in the section “11 cycles for 22 weeks.” BAL = bronchoalveolar lavage; IM = intramuscular; PFT = pulmonary function testing; SQ = subcutaneous.

Figure 2.

Airway resistance. Change in airway resistance in each animal over the course of exposure protocol. Filtered-air (FA) animals are compared with HDMA-treated animals. Over the course of the treatment protocol, HDMA-treated animal developed a significant increase in airway resistance as compared with FA animals (p = 0.0037). Individual animal values are plotted with boxes representing 50% of the values and the median line.

Figure 3.

(A) Airway remodeling. Hematoxylin-and-eosin–stained frozen sections from proximal (1, 3) to distal (5, 7) airway levels of FA- and HDMA-treated animals. HDMA-treated animals have an increase in number of inflammatory cells in the subepithelial mucosa, thicker basement membrane, and mucous cell hypertrophy. Bar = 20 μm. (B) Vascular density in bronchial mucosa. Distribution of von Willibrand factor–positive bronchial vessel in proximal (1, 3) to distal (5, 7) airway generations. Top row shows representative images from FA animals, bottom row shows HDMA animals. HDMA-treated animals have a greater number and density of blood vessels. Background autofluorescence is orange. Bar = 5 μm.

Figure 4.

Airway remodeling schematic. Split figure demonstrating the main features of airway remodeling seen in asthmatic rhesus macaques. In addition to mucous hyperplasia and a thickened basement membrane, blood vessels in the airway mucosa are shown in greater number and density. Random intersections of vessel surface area are referenced to random intersections of the epithelial basement membrane to provide a stereologic ratio of vessel surface–to–basement membrane surface.

Figure 5.

Morphometry. (A) Surface of endothelium to surface of the epithelial basement membrane. Random intersections of bronchial vessel as a ratio to intersections of epithelial basement membrane at each airway generation. Error bars designate variability within each group at that airway generation. Significant values represented by single asterisks and include p = 0.039 (airway generation 3), p = 0.025 (airway generation 7). (B) Surface of endothelium to the volume of interstitium determined by quantitating intersection of endothelium expressed in a ratio with random point density of interstitium at each airway generation. Values are means ± SE and represent variation of the group at each airway generation. Significant values are represented by double asterisks and include surface of endothelium to volume of interstitium, p = 0.024 (airway generation 3), p = 0.009 (airway generation 5), p = 0.012 (airway generation 7).

Figure 5.

Morphometry. (A) Surface of endothelium to surface of the epithelial basement membrane. Random intersections of bronchial vessel as a ratio to intersections of epithelial basement membrane at each airway generation. Error bars designate variability within each group at that airway generation. Significant values represented by single asterisks and include p = 0.039 (airway generation 3), p = 0.025 (airway generation 7). (B) Surface of endothelium to the volume of interstitium determined by quantitating intersection of endothelium expressed in a ratio with random point density of interstitium at each airway generation. Values are means ± SE and represent variation of the group at each airway generation. Significant values are represented by double asterisks and include surface of endothelium to volume of interstitium, p = 0.024 (airway generation 3), p = 0.009 (airway generation 5), p = 0.012 (airway generation 7).

Figure 6.

Airway level–specific vascular endothelial growth factor (VEGF) splice variant gene expression. Airway generation–specific gene expression of VEGF splice variants (A) 121, (B) 165, and (C) 189 normalized to control gene. VEGF₁₂₁ is significantly elevated at distal airway generation, p = 0.004 (*). VEGF₁₆₅ approached significance, p = 0.061, at the same distal airway level. Values are means ± SE.

Figure 6.

Airway level–specific vascular endothelial growth factor (VEGF) splice variant gene expression. Airway generation–specific gene expression of VEGF splice variants (A) 121, (B) 165, and (C) 189 normalized to control gene. VEGF₁₂₁ is significantly elevated at distal airway generation, p = 0.004 (*). VEGF₁₆₅ approached significance, p = 0.061, at the same distal airway level. Values are means ± SE.

Figure 6.

Airway level–specific vascular endothelial growth factor (VEGF) splice variant gene expression. Airway generation–specific gene expression of VEGF splice variants (A) 121, (B) 165, and (C) 189 normalized to control gene. VEGF₁₂₁ is significantly elevated at distal airway generation, p = 0.004 (*). VEGF₁₆₅ approached significance, p = 0.061, at the same distal airway level. Values are means ± SE.

Figure 7.

VEGF secretion from cultured monkey primary tracheal epithelial cells. VEGF protein secretion measured by ELISA in pg/ml from cultured tracheal epithelial cells in the presence of increasing HDMA concentration. * Different than all other treatment conditions (p < 0.05); ⁺ different than HDMA group (p < 0.05). Values are means ± SE.

Figure 8.

VEGF isoform gene expression in cultured monkey primary tracheal epithelial cells. Semiquantitative polymerase chain reaction (PCR) analysis of VEGF isoforms from tracheal epithelial cells cultures in presence of increasing HDMA. (A) Representative PCR reaction on 2% agarose gel. (B) Relative amount of each splice variant at increasing HDMA concentration. Relative quantity of VEGF₁₂₁ increased with HDMA exposure, p = 0.08. Values are means ± SE.

Figure 8.

VEGF isoform gene expression in cultured monkey primary tracheal epithelial cells. Semiquantitative polymerase chain reaction (PCR) analysis of VEGF isoforms from tracheal epithelial cells cultures in presence of increasing HDMA. (A) Representative PCR reaction on 2% agarose gel. (B) Relative amount of each splice variant at increasing HDMA concentration. Relative quantity of VEGF₁₂₁ increased with HDMA exposure, p = 0.08. Values are means ± SE.