The Effects of Tumstatin on Vascularity, Airway Inflammation and Lung Function in an Experimental Sheep Model of Chronic Asthma

Abstract

Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10(+)-20 μm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.

Figure 1. Lung segments used and timeline for procedures and measurements performed on the sheep.

(a) A sheep lung which has been labelled to show the different lung segments used for the different treatments. The left and right distal lung segments received challenges of solubilized house dust mite (HDM) (200 μg in 5 mL saline) weekly and treatment with either Tumstatin (150 μg in 5 mL saline) or saline (5 mL) twice weekly respectively. The right medial segment received an equivalent volume of saline to act as a control for HDM. (b) Shows the time-line for all treatments and procedures performed on the sheep.

Figure 2. Airway responsiveness to allergic and non-allergic stimuli at week 24.

(a) Percent change in airway peripheral airway resistance (R_p) 30 mins post-HDM from the resistance after saline. *p < 0.05. (b) Percent dose of methacholine required to increase R_p 100% from the resistance after saline (PC₁₀₀) 24 hours following HDM challenge. Mean + SEM (n = 7 sheep).

Figure 3. Leukocyte responses in the differentially treated lung segments.

Panel (a) shows the numbers of leukocytes recovered from BAL fluid at week 22 of the trial both before (0 hr), and 48 hrs after, house dust mite (HDM) or saline challenges to the different segments. *p < 0.05 48 hr vs 0 hr. The data in panels (b,c) were obtained from frozen sections of the differentially treated lung segments which were immunostained for CD8- and CD4-positive lymphocytes, eosinophils, and gamma delta-positive T cell receptor cells. Panel (b) shows leukocyte densities in the airway wall of the differenti lung segments. Panel (c) shows leukocyte densities in the lung parenchyma of the different lung segments. The results are expressed as means of the differentially treatment segments. *p < 0.05 HDM + saline vs vehicle saline, and HDM + tumstatin vs HDM + saline (n = 7 sheep).

Figure 4. Airway blood vessel density in the differentially treated lung segments.

Representative photomicrographs of blood vessels stained with Type IV Collagen are shown for (a) HDM + saline, and (b) HDM + tumstatin treated lung segments. (c) Shows the density of blood vessels in the airway lamina propria immuno-stained with Type IV Collagen (n = 7 sheep).

Figure 5. Density of different sized blood vessels in the airway lamina propria of differentially treated lung segments.

Blood vessels in frozen sections were immuno-labelled with a ColIV antibody and then sorted into four different size groups based on their diameters. The diameter ranges in the groups were 0–10 μm, 10⁺–20 μm, 20⁺–30 μm and 30⁺ μm. *p < 0.05 HDM + tumsatin treatment vs HDM + saline (n = 7 sheep).

Figure 6. VEGF expression in ASM.

Representative photomicrographs of airway sections immunostained with an antibody against VEGF for the HDM + saline treated lung segment (a), and the HDM + tumstatin treated lung segment (b). (c) Shows the mean intensity of VEGF immunostaining in individual sheep in the three differentially treated lung segments. *p < 0.05 (n = 7 sheep).