A Treatment with a Protease Inhibitor Recombinant from the Cattle Tick (Rhipicephalus Boophilus microplus) Ameliorates Emphysema in Mice

Abstract

AimsTo determine whether a serine protease inhibitor treatment can prevent or minimize emphysema in mice.MethodsC57BL/6 mice were subjected to porcine pancreatic elastase (PPE) nasal instillation to induce emphysema and were treated with a serine protease inhibitor (rBmTI-A) before (Protocol 1) and after (Protocol 2) emphysema development. In both protocols, we evaluated lung function to evaluate the airway resistance (Raw), tissue damping (Gtis) and tissue elastance (Htis). The inflammatory profile was analyzed in the bronchoalveolar lavage (BALF) and through the use of morphometry; we measured the mean linear intercept (Lm) (to verify alveolar enlargement), the volume proportion of collagen and elastic fibers, and the numbers of macrophages and metalloprotease 12 (MMP-12) positive cells in the parenchyma. We showed that at both time points, even after the emphysema was established, the rBmTI-A treatment was sufficient to reverse the loss of elastic recoil measured by Htis, the alveolar enlargement and the increase in the total number of cells in the BALF, with a primary decrease in the number of macrophages. Although, the treatment did not control the increase in macrophages in the lung parenchyma, it was sufficient to decrease the number of positive cells for MMP-12 and reduce the volume of collagen fibers, which was increased in PPE groups. These findings attest to the importance of MMP-12 in PPE-induced emphysema and suggest that this metalloprotease could be an effective therapeutic target.

Collagenase mRNA Overexpression and Decreased Extracellular Matrix Components Are Early Events in the Pathogenesis of Emphysema

“…After 21 days of PPE instillation we observed an increase in the proportion of elastin in mice lungs, which is in agreement with previous studies [ 22 , 25 ]. However, many studies indicated that despite the increased elastin content after emphysema development, these fibers are non-functional due to their molecular complexity, which renders repair processes of these fibers inefficient leading to impairment of lung function [ 16 , 47 ], as showed previous by our group after 21 days of the emphysema induction using this same experimental model [ 30 ].…”

“…Then, both lungs were fixed using 10% buffered formalin infused through the trachea at a constant pressure of 20 cm H 2 0 for 24 hours, embedded in paraffin and cut into 5 μm coronal sections. Lung tissue sections were stained with H&E for lung structure analysis [ 30 ]. For PCR analysis, lungs were carefully dissected from the mediastinum and immediately immersed in RNAlater stabilizer and RNA storage solution (Applied Biosystems Canada, Streetsville, ON, Canada) at room temperature, preserved overnight at 4°C and subsequently stored at -80°C until further analysis.…”

Peptidase inhibitors in tick physiology

“…BmTI-A does not change prothrombin and thrombin time, but increases the activated partial thromboplastin time (aPTT), and it has recently been suggested to inhibit angiogenesis in an in vitro vessel formation assay (Soares et al, 2016). In addition, the use of rBmTI-A as a therapy in elastase-induced experimental emphysema appears to minimize parenchymal lesions in mice, suggesting the potential application of this inhibitor in emphysema treatment (Lourenço et al, 2014). Other R. microplus Kunitz-type inhibitors include BmTI-6, a three-Kunitz-domain plasmin inhibitor expressed in the ovary and fat body ; Rhipicephalus microplus kallikrein (RmKK) inhibitor a two-Kunitz-domain inhibitor active against trypsin and tissue/pancreatic kallikrein (Abreu et al, 2014); and boophilin, an anticoagulant and antithrombotic two-Kunitz-domain inhibitor expressed in the midgut of fully engorged females, which inhibits thrombin, elastase, kallikrein, cathepsin G and plasmin (Macedo-Ribeiro et al, 2008.…”

O papel dos macrófagos M1 e M2 no enfisema pulmonar em modelos experimentais de indução por instilação de elastase e exposição à fumaça de cigarro

“…Entretanto, como já descrito por Lourenço e col. (56) , ambos os modelos demonstraram aumento do processo inflamatório com aumento da densidade de células positivas para MAC-2, MMP-12 e MMP-9.…”