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In situ Evidence of Collagen V and Interleukin-6/Interleukin-17 Activation in Vascular Remodeling of Experimental Pulmonary Hypertension

Batah S.S.a · Alda M.A.a · Rodrigues Lopes Roslindo Figueira R.a · Cruvinel H.R.b · Perdoná Rodrigues da Silva L.b · Machado-Rugolo J.b · Velosa A.P.c · Teodoro W.R.c · Balancin M.f · Silva P.L.d,e · Capelozzi V.L.f · Fabro A.T.a

Author affiliations

aDepartment of Pathology and Legal Medicine, Riberão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
bBotucatu Medical School, São Paulo State University, Botucatu, Brazil
cRheumatology Division of the Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
dLaboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Centro de Ciências da Saúde, Rio de Janeiro, Brazil
eNational Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil
fLaboratory of Histomorphometry and Lung Genomics, Faculty of Medicine, University of São Paulo, São Paulo, Brazil

Corresponding Author

Vera Luiza Capelozzi

Department of Pathology

Faculdade de Medicina da Universidade de São Paulo

Av. Dr. Arnaldo 455, Sala 1143, São Paulo, SP 01246-903 (Brazil)

vera.capelozzi@fm.usp.br

Keywords: Collagen type VCollagen type IMonocrotalinePulmonary arterial hypertensionImmunofluorescenceInterleukin-6Interleukin-17

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Pathobiology 2020;87:356–366
https://doi.org/10.1159/000510048

Abstract

Several studies have reported the pathophysiologic and molecular mechanisms responsible for pulmonary arterial hypertension (PAH). However, the in situ evidence of collagen V (Col V) and interleukin-17 (IL-17)/interleukin-6 (IL-6) activation in PAH has not been fully elucidated. We analyzed the effects of collagen I (Col I), Col V, IL-6, and IL-17 on vascular remodeling and hemodynamics and its possible mechanisms of action in monocrotaline (MCT)-induced PAH. Twenty male Wistar rats were randomly divided into two groups. In the PAH group, animals received MCT 60 mg/kg intraperitoneally, whereas the control group (CTRL) received saline. On day 21, the pulmonary blood pressure (PAP) and right ventricular systolic pressure (RVSP) were determined. Lung histology (smooth muscle cell proliferation [α-smooth muscle actin; α-SMA] and periadventitial fibrosis), immunofluorescence (Col I, Col V, and α-SMA), immunohistochemistry (IL-6, IL-17, and transforming growth factor-beta [TGF-β]), and transmission electron microscopy to detect fibronexus were evaluated. The RVSP (40 ± 2 vs. 24 ± 1 mm Hg, respectively; p < 0.0001), right ventricle hypertrophy index (65 ± 9 and 25 ± 5%, respectively; p < 0.0001), vascular periadventitial Col I and Col V, smooth muscle cell α-SMA+, fibronexus, IL-6, IL-17, and TGF-β were higher in the MCT group than in the CTRL group. In conclusion, our findings indicate in situ evidence of Col V and IL-6/IL-17 activation in vascular remodeling and suggest that increase of Col V may yield potential therapeutic targets for treating patients with PAH.

© 2020 S. Karger AG, Basel

Introduction

Pulmonary arterial hypertension (PAH) is a hemodynamic phenomenon characterized by increased pulmonary vascular resistance and increased pressure to maintain cardiac output, leading to right heart failure [1]. In PAH, the reduced pulmonary arterial compliance is due to extracellular matrix (ECM) remodeling of the pulmonary vascular system. Growing evidence suggests that the decrease in pulmonary arterial compliance and increased pulsatility play a critical role in the pathogenesis of PAH and are a cause rather than a consequence of distal small vessel proliferative vasculopathy [2]. Considering the prognostic importance of ECM and its causative role in the development and progression of distal small vessel proliferative vasculopathy, the ECM is an attractive target for developing novel therapies for PAH.

Collagens are the most abundant components of the ECM, representing a widespread distribution among tissues including pulmonary arteries [3]. They are categorized according to their common homology and function. Fibrillar collagens include collagen types I (Col I), III (Col III), and V (Col V), which represent the most abundant and widespread collagens in tissues. Col V is a minor collagen found primarily within the fibrils of the major lung collagens, such as Col I and Col III. Col V regulates collagen fiber assembly, geometry, strength, and linking stromal collagen to the basement membrane [4, 5]. Vascular injury may lead to the exposure of Col V, a normally sequestered antigen, allowing an autoimmune response activation [6]. The continuous autoimmune response may lead to unusual vascular remodeling, as evidenced in clinical and preclinical studies showing that increased levels of Col V correlate with fibrosis and disease progression [7-10]. Th17 cells, a subset of interleukin-17 (IL-17)-producing effector T cells, have also been implicated in the pathogenesis of multiple autoimmune diseases [11, 12]. Transforming growth factor-beta (TGF-β) is a strong inducer of IL-17 [13], and IL-17, in turn, may be involved in vascular fibrogenesis in TGF-β-dependent and independent pathways [14]. In addition, patients with idiopathic PAH exhibit increased interleukin-6 (IL-6) serum levels, a multifunctional proinflammatory cytokine associated with several autoimmune diseases, correlating with their prognoses [15, 16]. In agreement with these reports, lung-specific IL-6 transgenic mice showed spontaneous pulmonary hypertension and developed significantly overstated hypoxia-induced pulmonary hypertension (HPH) [17], whereas IL-6–deficient mice showed resistance to HPH [18]. Cytokine IL-6 initiates and orchestrates inflammatory cell infiltration [19], whereas proinflammatory IL-17 induces vascular oxidases and also dysfunction in eNOS (endothelial nitric oxide synthase) endothelium, vascular smooth muscle cells, and adventitial fibroblasts [20]. These events link vascular dysfunction to cardiovascular disorders, including atherosclerosis, hypertension, diabetes, and obesity [20].

Although several studies have been conducted and notable progress made in understanding the pathophysiologic and molecular mechanisms responsible for pulmonary hypertension, the in situ evidence of Col V and IL-6/IL-17 activation in PAH has not been fully elucidated. Therefore, identification of factors that regulate pulmonary vascular remodeling could lead to new therapies for PAH treatment. The present study uses a rat model of monocrotaline (MCT)-induced PAH to investigate Col V, IL-6, and IL-17 effects on vascular histology and hemodynamics and to attempt to elucidate the possible mechanisms in experimental PAH.

Methods

Ethics Statements

This study was approved by the Institutional Animal Care and Use Committee of the Health Sciences Centre, State University School of Medicine, Botucatu (CEUA 1026-2013), and registered by the Brazilian National Council for Animal Experimentation Control. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the US National Academy of Sciences Guide for the Care and Use of Laboratory Animals. The present study followed the ARRIVE guidelines for reporting of animal research [21].

Animal Preparation and Experimental Protocol

Male Wistar rats (weight 235 ± 21 g) were randomized into two groups: MCT-induced PAH (MCT, n = 20), with 60 mg/kg of MCT intraperitoneally (C2401; Sigma Chemical Co., St Louis, MO, USA), and a control group (CTRL, n = 20), with a similar volume of saline solution intraperitoneally.

Hemodynamic Measurements

Hemodynamic measurements were taken in all animals after 21 days. Pulmonary blood pressure (PAP) was measured using the standard technique described by Rabinovitch [22]. Following intraperitoneal administration of anesthesia with 0.3 mg/kg of aqueous thiazine solution (Rompum, Bayer, Brazil) and 10 mg/kg of ketamine hydrochloride (Ketalar, Park-Davis), orotracheal intubation was performed with controlled mechanical ventilation (Harvard Rodent Ventilator, USA). The internal jugular vein was dissected for introduction of a catheter and transducer (ADInstruments), and then connected to a pressure monitor (PowerLab Data Acquisition System, ADInstruments). The catheter was introduced into the right ventricle and pulmonary trunk under pressure tracing guidance. After 15 min of stabilization, PAP was measured for a further 15 min. The average PAP (mPAP) was calculated by digital integration. At the end of the experiment, the animals were euthanized by a lethal dose of anesthetic (100 mg/kg ketamine hydrochloride).

Assessment of Right Ventricular Hypertrophy Index

After hemodynamic measurements, the heart and lung were removed. The heart, aorta, pulmonary artery trunk, and atria were dissected. The right ventricular wall was separated from the septum (S), and the septum and left ventricular wall (LV) weight were determined. The right ventricular hypertrophy index was expressed by the formula: VD/(LV + S) × 100.

Lung Preparation

After the lethal dose of anesthetic (100 mg/kg ketamine hydrochloride), the inferior vena cava was clamped and the aorta was cut and opened. A Silastic cannula was introduced into the right atrium for nitroprusside infusion (1 mg/mL; Sigma) at 18 mm Hg for 10 min. Then, under 25 mm Hg pressure, the main bronchus from the left lower lobe was tied, separated, and stored in 4% glutaraldehyde, and the right lung was fixed with dripped 10% buffered formalin followed by 2 h of fixation in formalin equivalent to one tenth of the total lung volume. The lung was sampled using the isotropic uniform random sampling technique recommended by the ATS/ERS standards for quantitative assessment of pulmonary structures, allowing greater accuracy and precision [23].

Histology, Immunofluorescence, Immunohistochemistry, and Morphometry

The lungs were again fixed in buffered 10% formalin for 24 h at room temperature (RT) and entirely sectioned. All sections from each individual rat were paraffin embedded, and consecutive 3-µm-thick sections of each sample were stained with hematoxylin and eosin (HE) for histological analysis.

The Cavaliere sampling sequence [24] was conducted to estimate lung vessels volume, followed by point estimation using a grid to obtain the fractional volume. Finally, an intersection scoring system was used to estimate the volume of interest using the internal elastic membrane of the vessels as a reference. This method avoids bias and normalizes the size of the arteries [24].

The vessel area was defined as the media area between the inner and outer elastic laminae and the adventitia as the area between the outer elastic lamina and the edge of the adventitia connective tissue. According to the vessel size, which is defined by the shortest distance between the internal elastic laminae, they were classified as follows: small arteries (30–100 μm), medium arteries (101–200 μm), and large arteries (201–500 μm). Twenty arteries from each animal were analyzed for each type of vessel. The extent of smooth muscle hypertrophy in vessels was evaluated as: muscularized (double elastic membrane in more than half of the vessels), partially muscularized (double elastic membrane in less than half of the vessels), and not muscularized (only one elastic membrane).

The total collagenous fibers in the periadventitial layer of small and median arteries were identified by the picrosirius-polarization method according to previous work [25-28]. Type I (Col I), III (Col III), and V (Col V) collagen fibers were characterized by immunofluorescence according to our laboratory protocol [28-31]. Briefly, lung sections (3–4 μm) were dewaxed in xylol, hydrated in graded ethanol, and incubated overnight at 4°C with rabbit polyclonal anti-human Col I (1:700, Rockland), anti-human Col V (1:1.000, Rockland), and anti-α-SMA (smooth muscle actin; 1:500, Abcam) antibodies diluted in phosphate-buffered saline. The slides were stained in green color with an Alexa 488-conjugated anti-mouse immunoglobulin G (IgG; Invitrogen, Eugene, OR, USA), and the nuclei were counterstained with 0.4 mM/mL 4′,6-diamidino-2-phenylindole, dihydrochloride (Molecular ProbesTM, Invitrogen, Eugene, OR, USA) for 15 min at RT. The specimens were observed using a laser-scanning microscope (Zeiss LSM 510 META/UV, Germany).

The area occupied by total collagen, Col I, Col III, and muscularization α-SMA+ was determined in media and adventitial layers at ×400 magnification in 20 random fields, with the aid of a digital analysis system and specific software (Image-Pro Plus 4.1 for Windows; Media Cybernetics, Silver Spring, MD, USA). The images were generated by a microscope (Axioplan, Zeiss) connected to a camera (Trinitron CCD, Sony, Tokyo, Japan), fed into a computer through a frame grabber (Oculus TCX, Coreco, St. Laurent, QC, Canada) for offline processing. The thresholds for collagen fibers were established after enhancing the contrast up to a point at which the fibers were easily identified as birefringent (collagen) bands and kept constant during the measurements. The area occupied by fibers was determined by digital densitometric recognition. The results were expressed as the amount of total collagen fibers, Col I, Col III, Col V, and α-SMA per total media and adventitia area of each caliber vessel (µm2/µm).

To evaluate inflammatory and fibrotic mediators, immunohistochemistry was performed according to our laboratory protocol [19, 32-34]. Succinctly, lung sections were dewaxed in xylene, hydrated in graded ethanol, rehydrated in deionized water, and incubated overnight at 4°C with the following primary antibodies: anti-IL-6 (SC-152, 1:100 dilution), anti-IL-17 (Sc-7927, dilution 1:100), and anti-TGF-β1 (Sc-146, 1:500 dilution). After three washes, the sections were incubated with an anti-rabbit or anti-rat horseradish peroxidase-conjugated secondary antibody (Vectastain, Vector Labs Inc., Peterborough, UK) for 30 min at RT. The peroxidase reaction was developed for 10 min with the use of diaminobenzidine as the chromogen (Impact DAB, Vector Labs Inc.) and blocked with deionized water. Substitution of the primary antibody with a rabbit- or rat-unrelated primary polyclonal serum served as a negative control. The positive controls used were as follows: (1) IL-6 showing staining in the cytoplasm of paraffin-embedded spleen tissue, (2) IL-17 showing staining in the cytoplasm of paraffin-embedded small intestine tissue, and (3) TGF-1 showing staining in the cytoplasm of paraffin-embedded intestine tissue.

The immunostained cells in lung vessels were quantified by histomorphometry. Twenty arteries from each animal were analyzed for each type of vessel in the proximal and distal lung parenchyma. The measurements were made with a 100-point and 50 straight grid with a known area (103 µm2 at a ×400 magnification) attached to the ocular part of the microscope [35, 36]. At ×400 magnification, the number of positive immunostained cells in each vessel was calculated according to the number of points hitting positive cells for specific antibodies as a proportion of the total vessel area. The results were expressed as the number of positive cells/mm2 of vessel area.

Electron Microscopy

The left lower lobe fixed in 4% glutaraldehyde was used for electron microscopy. The specimens were washed overnight in 0.9% saline containing uranyl and sucrose and were Epon embedded. For each case, three or five lung fragments were sectioned at 1 μm and selected by light microscopy. Acceptable sections were those that represent the histological pattern of the lung identified in most fragments. Their respective Epon-embedded blocks were sectioned at 55–60 nm, stained with uranyl acetate and lead citrate, and examined with a JEOL JEM-1010 electron microscope.

Statistical Analysis

The samples obtained from the quantitative procedure were expressed as the mean ± standard error of the mean (SE) and were evaluated according to the distribution of morphological changes in vessels from the MCT and CTRL groups. The descriptive analysis was demonstrated by the Student t test, which was applied to verify the differences between the groups. When necessary, the Tukey test was used to identify differences between measurements. A p value <0.05 was considered statistically significant.

Results

Hemodynamic Measurements

On day 21, MCT animals exhibited higher right ventricular systolic pressure (RVSP) than did CTRL animals (40 ± 2 and 24 ± 1 mm Hg, respectively; p < 0.0001). Likewise, the right ventricle hypertrophy index was higher in MCT animals compared with CTRL animals (65 ± 9 and 25 ± 5% respectively; p < 0.0001).

MCT Pulmonary Vascular Injury

Twenty-one days after MCT, histologic examination of the pulmonary small arteries (50 µm) was performed by HE staining, which showed smooth muscle hypertrophy in the media layer and subocclusion of the lumen (Fig. 1a, b). Interestingly, the loose connective tissue around the adventitial layer is predominant in 50- to 100-µm-diameter vessels (Fig. 1b), coinciding with a significant increase of adventitial layer thickening (p < 0.05; Fig. 1c). Furthermore, a critical increase in orange-red birefringence of thick collagen fiber deposition in the periadventitial layer of small vessels was found in the MCT group compared with the CTR group (Fig. 1d, e), coinciding with adventitial thickening of the small arteries (p < 0.05; Fig. 1f).

Fig. 1.

Effects of MCT on lung vascular architecture. Wistar rats receiving MCT 60 mg/kg intraperitoneally. Histological changes were demonstrated by HE (a–b) and Picro sirius (d–e). Two independent individuals measured arterial fractional volumes by the Cavaliere sampling sequence. Increased smooth muscle hypertrophy (single arrows) and periadventitial thickening (two-headed arrows) in arteries measuring 50 and 50–100 µm were evident in MCT animals (a–c). Rats receiving MCT exhibited increased orange-red birefringence of periadventitial total collagen compared with controls (d–f). Values are the means ± SE (n = 20). * p < 0.05. Alv, alveolus; Art, artery.

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Immunofluorescence staining of control lungs showed green birefringence of Col I fibers and weak green birefringence of α-SMA loosely arranged around small arteries (50 µm) and along the adventitial layer, assuming a uniform distribution, enhancing pulmonary vascular architecture (Fig. 2a, b), and coinciding with normal thickness of the periadventitial layer (Fig. 2g, p < 0.05) and the smooth muscle layer (Fig. 2h; p < 0.05). In contrast, on day 21 after MCT, significantly strong green birefringence respective of thick Col I fibers and α-SMA were tightly packed around small vessels and along the adventitial layer, modifying the usual histoarchitecture of pulmonary vessels when compared with the control (Fig. 2d, e, g, h; p < 0.05). The smooth muscle proliferation extended outside the adventitial layer, promoting muscularization of alveolar ducts (Fig. 2e). Ultrastructurally, Col I fibers were increased and strongly disarranged, tending to accumulate around α-SMA+ myofibroblasts. In addition, the presence of fibronexus (p < 0.05; Fig. 2f, i) was noted, contrasting with its absence in control lungs (Fig. 2c, i).

Fig. 2.

Changes in Col I fibers, α-SMA, and fibronexus in vascular injury of the lung following MCT 60 mg/kg intraperitoneally. Immunofluorescence was performed to characterize Col I fibers and α-SMA in the small arteries of lung (a, b, d, e). Transmission electron microscopy evaluated the fractional volume of fibronexus (c, f). Two independent individuals measured Col I, α-SMA, and fibronexus volumes in small arteries by Image-Pro Plus. Rats receiving MCT exhibited increased green birefringence (arrows) respective of deposition of Col I fibers (d, g; p < 0.05) and α-SMA (e, h; p < 0.05). Note the presence of fibronexus (adjacent arrows) and Col I fibers (f, i; p < 0.05) in the small arteries compared with the absence in controls (c). Values are the means ± SE (n = 20). * p < 0.05. Alv, alveolus; Art, artery; MF, myofibroblast.

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Col V analysis in the CTRL group showed weak green birefringence of loose fibers in a homogeneous, linear distribution around the vessels (Fig. 3a), consistent with a normal pulmonary vascular architecture. In contrast, after 21 days, MCT exhibited a significant strong green birefringence of thick Col V fibers, assuming an irregular and micronodular distribution involving the adventitial layer of small arteries in the MCT group when compared with the controls (Fig. 3a–c; p < 0.05).

Fig. 3.

Changes in Col V fibers in vascular injury of the lung following MCT 60 mg/kg intraperitoneally. Immunofluorescence was performed to characterize Col V fibers. Two independent individuals measured Col V fractional areas in small arteries by Image-Pro Plus. Rats receiving MCT exhibited increased green birefringence (arrows) of Col V fiber deposition (b, c; p < 0.05) in the small arteries compared with controls. Values are the means ± SE (n = 20). * p < 0.05. Art, artery; Alv, alveolus.

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Compared with the CTRL group, immunophenotypes of TGF-β (Fig. 4a–c), IL-6 (Fig. 4d–f), and IL-17 (Fig. 4g–i) were significantly overexpressed in MCT at 21 days (p < 0.05). Furthermore, we found that Col V was statistically associated with TGF-β (R = 0.70; p < 0.05), IL-6 (R = 0.65; p < 0.05), and IL-17 (R = 0.75; p < 0.05).

Fig. 4.

Immunohistochemistry in vascular injury of the lung following MCT 60 mg/kg intraperitoneally. Immunohistochemical analysis evaluated the lung of rats receiving the treatment with MCT. Positive cell staining with periadventitial markers IL-6 and IL-17, and TGF-β are indicated by brown color (arrows; b, e, h). Two independent individuals measured the fractional areas of IL-6, IL-17, and TGF-β by histomorphometry. Rats receiving MCT exhibited increased expression of IL-6 (b, c; p < 0.05), IL-17 (e, f; p < 0.05), and TGF-β (h, i; p < 0.05) compared with controls (a, d, g). Values are the means ± SE (n = 20). ** p < 0.01. Alv, alveolus; Art, artery.

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Discussion

In the present study, we aimed to evaluate in situ evidence of Col V and IL-17/IL-6 activation in experimental PAH. Therefore, the identification of factors that regulate pulmonary vascular remodeling could lead to new therapies to treat PAH. Under the conditions of this study, in situ evidence of Col V, IL-6, and IL-17 activation on vascular ECM presented a variety of associated effects on MCT-induced PAH, including: (1) increased RVSP; (2) pulmonary artery smooth muscle hypertrophy and hyperplasia lead to increased medial thickness in small pulmonary arteries and muscularization of alveolar duct walls; (3) expansion of the ECM in media and periadventitial layers of the pulmonary vascular wall lead to vascular fibrosis; (4) increased Col I and Col V; (5) increased TGF-β expression, and (6) increased IL-6 inflammatory and IL-17 profibrotic markers in media and the adventitial layer of pulmonary small arteries. For the past 3 decades, two rodent models have been central for the investigation of human pulmonary hypertension: the chronic hypoxia exposure model and the MCT lung injury model [19, 37, 38]. Although the mechanisms of hypoxia-induced vascular remodeling are understood to some degree, the complex plexiform lesions found in human patients with severe PAH does not develop in the rodent model [39]. The MCT rat model remains an often-investigated model of PAH, since it offers technical simplicity, reproducibility, and low cost compared with other PAH models [40]. In this line, we observed several pathophysiological features associated with PAH in animals exposed to MCT, such as high RVSP and high right ventricle hypertrophy index compared with CTRL animals. Histologic examination of the pulmonary small arteries showed medial hypertrophy and smooth muscle cell thickening. Interestingly, the loose connective tissue around the adventitial layer was predominant in 50- to 100-µm-diameter vessels coinciding with increased adventitial layer thickening. All these findings represent specific features in PAH [41, 42]. The lumen reduction by proliferation of vascular endothelium combined with constriction of the arteriole due to remodeling and periadventitial fibrosis results in low pulmonary arterial blood pressure and a decrease in the cardiac output. Furthermore, we observed a critical increase of total collagen deposition in small vessels in MCT animals coinciding with adventitial thickening of small vessels. In fact, we found after MCT (day 21) that the thick Col I fibers were significantly enlarged and tightly packed around the vessels and along the adventitial layer, modifying the usual histoarchitecture of pulmonary vessels. Ultrastructurally, these strongly disarranged enlarged fibers tend to accumulate around myofibroblasts, positive smooth muscle actin, and fibronexus, a cell surface specialization consisting of intracellular actin filaments and extracellular fibronectin filaments associated with sub-plasmalemmal plaque material. The fibronexus represents an intercellular junction between myofibroblasts, that is, a device that provides the contact between myofibroblasts and ECM, mediating continuity of intracellular contractile filaments and ECM proteins [43].

PAH is associated with autoimmunity and inflammation, currently recognized as critical contributors to its pathogenesis [44]. In this current work, we found increased Col V, which assumed an irregular and micronodular distribution involving the small arteries adventitial layer in the MCT group. Weber and Wilkes [6] reported that lung injury may lead to the exposure of this normally sequestered antigen, enabling an autoimmune response activation. In fact, clinical and preclinical studies show that increased levels of Col V correlate with fibrosis and disease progression, indicating that a continuous autoimmune response may lead to unusual vascular remodeling [7-10]. In this scenario, we found that increased Col V was associated with higher expression of profibrotic cytokines, such as IL-17 and TGF-β. Previous work has shown that the pathogenesis of several autoimmune diseases has also been implicated to Th17 cells, a subset of IL-17-producing effector T cells. Vittal et al. [8] reported in murine orthotopic lung transplants a feed-forward loop between IL-17 and TGF-β, leading them to assume that Col V and epithelial repair is associated with lung fibrosis. Subsequent studies done by Fabro et al. [45] showed that IL-17 interacts with the expression of Col V in the late state of experimental pulmonary fibrosis. IL-6 is a multifunctional proinflammatory cytokine that is also associated with several autoimmune diseases [16]. In Figure 5, we suggest graphically the mechanism involved in experimental PAH according to evidence of Col V and IL-17 activation. Iwanami et al. [46] showed that proinflammatory cytokines, such as IL-6, produced by T cells, lymphocytes B cells, and macrophages, may be responsible for hyperproliferation of pulmonary artery endothelial cells, smooth muscle cells, and adventitial thickening. In agreement with Iwanami et al. [46], in the present study we observed an increase in inflammatory markers, including IL-6 expression in the adventitial layer, as in previous preclinical studies using MCT in PAH animals. IL-6 is a key factor that facilitates a shift in pericytes from the quiescent state into their contractile, hyperproliferative state [47], which would increase pulmonary vascular resistance in PAH. Although we did not evaluate the precise mechanism of mitigation of the inflammatory process, it may rely on inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway [38]. By modulating the IL-6 expression, MCT may maximize the intense proliferation observed in PAH.

Fig. 5.

Graphical representation of the possible mechanism involved in in situ vascular remodeling in PAH.

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The limitations of the present study must be taken into account. First, MCT-induced PAH is not able to mimic all of the complex features observed in human PAH. Nevertheless, it is a well-established model of moderately severe PAH [48] and has been widely used not only to elucidate its pathophysiological mechanisms [49, 50], but also to propose new therapeutic interventions [51]. Second, we chose to analyze the roles of IL-6 and IL-17 cytokines specifically because these mediators are associated with Col V [52], as is shown in previous studies with the MCT experimental model [16, 53] and in human patients with PAH [44]. However, we cannot rule out the role of other growth factors. Third, while the idea that Col V may play a role in activating immune/inflammatory processes in PAH is exciting, our current studies only support a relationship (one time point, no interventions). Fourth, extensive time course analysis would need to be undertaken along with interventions to demonstrate cause-effect relationships. Fifth, the use of transgenic mouse models might be helpful in this regard. In conclusion, our findings indicate that the in situ evidence of Col V and IL-6/IL-17 activation is associated with vascular adventitial remodeling, suggesting that increased Col V may yield potential therapeutic targets for treating patients with PAH.

Acknowledgements

We are grateful to Ms. Esmeralda Miristeni Eher and Ms. Sandra de Morais Fernezlian for their expertise on immunohistochemical protocols.

Statement of Ethics

This study was approved by the Institutional Animal Care and Use Committee of the Health Sciences Centre, State University School of Medicine, Botucatu (CEUA 1026-2013), and registered by the Brazilian National Council for Animal Experimentation Control. Further details are provided in the text.

Conflict of Interest Statement

The authors declare that they do not have any conflicts of interest.

Funding Sources

The research reported in this publication was supported in part by the São Paulo Research Foundation (FAPESP; 2018/20403-6) and the National Council for Scientific and Technological Development (CNPq-483005/2012-6).


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  15. Soon E, Holmes AM, Treacy CM, Doughty NJ, Southgate L, Machado RD, et al. Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension. Circulation. 2010 Aug;122(9):920–7.
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  16. Hashimoto-Kataoka T, Hosen N, Sonobe T, Arita Y, Yasui T, Masaki T, et al. Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension. Proc Natl Acad Sci USA. 2015 May;112(20):E2677–86.
    External Resources
  17. Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, Waxman AB. Interleukin-6 overexpression induces pulmonary hypertension. Circ Res. 2009 Jan;104(2):236-44.
    External Resources
  18. Savale L, Tu L, Rideau D, Izziki M, Maitre B, Adnot S, et al. Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice. Respir Res. 2009 Jan;10(1):6.
    External Resources
  19. de Mendonça L, Felix NS, Blanco NG, Da Silva JS, Ferreira TP, Abreu SC, et al. Mesenchymal stromal cell therapy reduces lung inflammation and vascular remodeling and improves hemodynamics in experimental pulmonary arterial hypertension. Stem Cell Res Ther. 2017 Oct;8(1):220.
    External Resources
  20. Nosalski R, Guzik TJ. Perivascular adipose tissue inflammation in vascular disease. Br J Pharmacol. 2017 Oct;174(20):3496–513.
    External Resources
  21. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010 Jun;8(6):e1000412.
    External Resources
  22. Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest. 2008 Jul;118(7):2372–9.
    External Resources
  23. Hsia CC, Hyde DM, Ochs M, Weibel ER; ATS/ERS Joint Task Force on Quantitative Assessment of Lung Structure. An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure. Am J Respir Crit Care Med. 2010 Feb;181(4):394–418.
    External Resources
  24. Cavaliere F, Cina A, Biasucci D, Costa R, Soave M, Gargaruti R, et al. Sonographic assessment of abdominal vein dimensional and hemodynamic changes induced in human volunteers by a model of abdominal hypertension. Crit Care Med. 2011 Feb;39(2):344–8.
    External Resources
  25. Montes GS. Structural biology of the fibres of the collagenous and elastic systems. Cell Biol Int. 1996 Jan;20(1):15–27.
    External Resources
  26. Satomi E, Teodoro WR, Parra ER, Fernandes TD, Velosa AP, Capelozzi VL, et al. Changes in histoanatomical distribution of types I, III and V collagen promote adaptative remodeling in posterior tibial tendon rupture. Clinics. 2008 Feb;63(1):9–14.
    External Resources
  27. Godoy-Santos AL, Ranzoni L, Teodoro WR, Capelozzi V, Giglio P, Fernandes TD, et al. Increased cytokine levels and histological changes in cartilage, synovial cells and synovial fluid after malleolar fractures. Injury. 2017 Oct;48 Suppl 4:S27–33.
    External Resources
  28. Diniz-Fernandes T, Godoy-Santos AL, Santos MC, Pontin P, Pereira CA, Jardim YJ, et al. Matrix metalloproteinase-1 (MMP-1) and (MMP-8) gene polymorphisms promote increase and remodeling of the collagen III and V in posterior tibial tendinopathy. Histol Histopathol. 2018 Sep;33(9):929–36.
    External Resources
  29. Martins V, Teodoro WR, Velosa AP, Andrade P, Farhat C, Fabro AT, et al. Butylated hydroxytoluene induces type-V collagen and overexpression of remodeling genes/proteins in experimental lung fibrosis. Histol Histopathol. 2018 Oct;33(10):1111–23.
    External Resources
  30. Sant'Ana PG, Batah SS, Leao PS, Teodoro WR, de Souza SLB, Ferreira Mota GA, et al. Heart remodeling produced by aortic stenosis promotes cardiomyocyte apoptosis mediated by collagen V imbalance. Pathophysiology. 2018 Dec;25(4):373-79.
    External Resources
  31. Balancin ML, Teodoro WR, Farhat C, de Miranda TJ, Assato AK, de Souza Silva NA, et al. An integrative histopathologic clustering model based on immuno-matrix elements to predict the risk of death in malignant mesothelioma. Cancer Med. 2020 Jul;9(13):4836–49.
    External Resources
  32. Leite-Junior JH, Garcia CS, Souza-Fernandes AB, Silva PL, Ornellas DS, Larangeira AP, et al. Methylprednisolone improves lung mechanics and reduces the inflammatory response in pulmonary but not in extrapulmonary mild acute lung injury in mice. Crit Care Med. 2008 Sep;36(9):2621–8.
    External Resources
  33. de Oliveira MV, Rocha NN, Santos RS, Rocco MR, de Magalhães RF, Silva JD, et al. Endotoxin-induced emphysema exacerbation: a novel model of chronic obstructive pulmonary disease exacerbations causing cardiopulmonary impairment and diaphragm dysfunction. Front Physiol. 2019 May;10:664.
    External Resources
  34. Felix RG, Bovolato AL, Cotrim OS, Leao PD, Batah SS, Golim MA, et al. Adipose-derived stem cells and adipose-derived stem cell- conditioned medium modulate in situ imbalance between collagen I- and collagen V-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis. Histol Histopathol. 2020;35(3):289–301.
    External Resources
  35. Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS. 1988 May;96(5):379–94.
    External Resources
  36. Weibel ER. Lung morphometry: the link between structure and function. Cell Tissue Res. 2017 Mar;367(3):413–26.
    External Resources
  37. Meyrick B, Gamble W, Reid L. Development of Crotalaria pulmonary hypertension: hemodynamic and structural study. Am J Physiol. 1980 Nov;239(5):H692–702.
    External Resources
  38. Nogueira-Ferreira R, Vitorino R, Ferreira R, Henriques-Coelho T. Exploring the monocrotaline animal model for the study of pulmonary arterial hypertension: a network approach. Pulm Pharmacol Ther. 2015 Dec;35:8–16.
    External Resources
  39. Voelkel NF, Tuder RM. Hypoxia-induced pulmonary vascular remodeling: a model for what human disease? J Clin Invest. 2000 Sep;106(6):733–8.
    External Resources
  40. Gomez-Arroyo JG, Farkas L, Alhussaini AA, Farkas D, Kraskauskas D, Voelkel NF, et al. The monocrotaline model of pulmonary hypertension in perspective. Am J Physiol Lung Cell Mol Physiol. 2012 Feb;302(4):L363–9.
    External Resources
  41. Pietra GG, Capron F, Stewart S, Leone O, Humbert M, Robbins IM, et al. Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol. 2004 Jun;43(12 Suppl S):25S–32S.
    External Resources
  42. Jonigk D, Golpon H, Bockmeyer CL, Maegel L, Hoeper MM, Gottlieb J, et al. Plexiform lesions in pulmonary arterial hypertension composition, architecture, and microenvironment. Am J Pathol. 2011 Jul;179(1):167–79.
    External Resources
  43. Eyden BP. Brief review of the fibronexus and its significance for myofibroblastic differentiation and tumor diagnosis. Ultrastruct Pathol. 1993 Nov-Dec;17(6):611–22.
    External Resources
  44. Schermuly RT, Ghofrani HA, Wilkins MR, Grimminger F. Mechanisms of disease: pulmonary arterial hypertension. Nat Rev Cardiol. 2011 Jun;8(8):443–55.
    External Resources
  45. Fabro AT, da Silva PH, Zocolaro WS, de Almeida MS, Rangel MP, de Oliveira CC, et al. The Th17 pathway in the peripheral lung microenvironment interacts with expression of collagen V in the late state of experimental pulmonary fibrosis. Immunobiology. 2015 Jan;220(1):124–35.
    External Resources
  46. Iwanami K, Matsumoto I, Tanaka-Watanabe Y, Inoue A, Mihara M, Ohsugi Y, et al. Crucial role of the interleukin-6/interleukin-17 cytokine axis in the induction of arthritis by glucose-6-phosphate isomerase. Arthritis Rheum. 2008 Mar;58(3):754–63.
    External Resources
  47. Ricard N, Tu L, Le Hiress M, Huertas A, Phan C, Thuillet R, et al. Increased pericyte coverage mediated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth muscle-like cells in pulmonary hypertension. Circulation. 2014 Apr;129(15):1586–97.
    External Resources
  48. Maarman G, Lecour S, Butrous G, Thienemann F, Sliwa K. A comprehensive review: the evolution of animal models in pulmonary hypertension research; are we there yet? Pulm Circ. 2013 Dec;3(4):739–56.
    External Resources
  49. Arcot SS, Lipke DW, Gillespie MN, Olson JW. Alterations of growth factor transcripts in rat lungs during development of monocrotaline-induced pulmonary hypertension. Biochem Pharmacol. 1993 Sep;46(6):1086–91.
    External Resources
  50. Morty RE, Nejman B, Kwapiszewska G, Hecker M, Zakrzewicz A, Kouri FM, et al. Dysregulated bone morphogenetic protein signaling in monocrotaline-induced pulmonary arterial hypertension. Arterioscler Thromb Vasc Biol. 2007 May;27(5):1072–8.
    External Resources
  51. Alencar AK, Pereira SL, Montagnoli TL, Maia RC, Kümmerle AE, Landgraf SS, et al. Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats. Br J Pharmacol. 2013 Jul;169(5):953–62.
    External Resources
  52. Dart ML, Jankowska-Gan E, Huang G, Roenneburg DA, Keller MR, Torrealba JR, et al. Interleukin-17-dependent autoimmunity to collagen type V in atherosclerosis. Circ Res. 2010 Oct;107(9):1106–16.
    External Resources
  53. Pang L, Qi J, Gao Y, Jin H, Du J. Adrenomedullin alleviates pulmonary artery collagen accumulation in rats with pulmonary hypertension induced by high blood flow. Peptides. 2014 Apr;54:101–7.
    External Resources

Author Contacts

Vera Luiza Capelozzi

Department of Pathology

Faculdade de Medicina da Universidade de São Paulo

Av. Dr. Arnaldo 455, Sala 1143, São Paulo, SP 01246-903 (Brazil)

vera.capelozzi@fm.usp.br

Article / Publication Details

First-Page Preview
Abstract of Research Article

Received: February 14, 2020
Accepted: July 09, 2020
Published online: October 23, 2020
Issue release date: December 2020

Number of Print Pages: 11
Number of Figures: 5
Number of Tables: 0

ISSN: 1015-2008 (Print)
eISSN: 1423-0291 (Online)

For additional information: https://www.karger.com/PAT

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References

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  8. Vittal R, Fan L, Greenspan DS, Mickler EA, Gopalakrishnan B, Gu H, et al. IL-17 induces type V collagen overexpression and EMT via TGF-β-dependent pathways in obliterative bronchiolitis. Am J Physiol Lung Cell Mol Physiol. 2013 Mar;304(6):L401–14.
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  10. Lei GS, Kline HL, Lee CH, Wilkes DS, Zhang C. Regulation of Collagen V Expression and Epithelial-Mesenchymal Transition by miR-185 and miR-186 during Idiopathic Pulmonary Fibrosis. Am J Pathol. 2016 Sep;186(9):2310–6.
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  12. Nakano K, Yamaoka K, Hanami K, Saito K, Sasaguri Y, Yanagihara N, et al. Dopamine induces IL-6-dependent IL-17 production via D1-like receptor on CD4 naive T cells and D1-like receptor antagonist SCH-23390 inhibits cartilage destruction in a human rheumatoid arthritis/SCID mouse chimera model. J Immunol. 2011 Mar;186(6):3745–52.
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  13. Mangan PR, Harrington LE, O’Quinn DB, Helms WS, Bullard DC, Elson CO, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature. 2006 May;441(7090):231–4.
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  14. Mi S, Li Z, Yang HZ, Liu H, Wang JP, Ma YG, et al. Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-beta1-dependent and -independent mechanisms. J Immunol. 2011 Sep;187(6):3003–14.
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  15. Soon E, Holmes AM, Treacy CM, Doughty NJ, Southgate L, Machado RD, et al. Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension. Circulation. 2010 Aug;122(9):920–7.
    External Resources
  16. Hashimoto-Kataoka T, Hosen N, Sonobe T, Arita Y, Yasui T, Masaki T, et al. Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension. Proc Natl Acad Sci USA. 2015 May;112(20):E2677–86.
    External Resources
  17. Steiner MK, Syrkina OL, Kolliputi N, Mark EJ, Hales CA, Waxman AB. Interleukin-6 overexpression induces pulmonary hypertension. Circ Res. 2009 Jan;104(2):236-44.
    External Resources
  18. Savale L, Tu L, Rideau D, Izziki M, Maitre B, Adnot S, et al. Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice. Respir Res. 2009 Jan;10(1):6.
    External Resources
  19. de Mendonça L, Felix NS, Blanco NG, Da Silva JS, Ferreira TP, Abreu SC, et al. Mesenchymal stromal cell therapy reduces lung inflammation and vascular remodeling and improves hemodynamics in experimental pulmonary arterial hypertension. Stem Cell Res Ther. 2017 Oct;8(1):220.
    External Resources
  20. Nosalski R, Guzik TJ. Perivascular adipose tissue inflammation in vascular disease. Br J Pharmacol. 2017 Oct;174(20):3496–513.
    External Resources
  21. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010 Jun;8(6):e1000412.
    External Resources
  22. Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest. 2008 Jul;118(7):2372–9.
    External Resources
  23. Hsia CC, Hyde DM, Ochs M, Weibel ER; ATS/ERS Joint Task Force on Quantitative Assessment of Lung Structure. An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure. Am J Respir Crit Care Med. 2010 Feb;181(4):394–418.
    External Resources
  24. Cavaliere F, Cina A, Biasucci D, Costa R, Soave M, Gargaruti R, et al. Sonographic assessment of abdominal vein dimensional and hemodynamic changes induced in human volunteers by a model of abdominal hypertension. Crit Care Med. 2011 Feb;39(2):344–8.
    External Resources
  25. Montes GS. Structural biology of the fibres of the collagenous and elastic systems. Cell Biol Int. 1996 Jan;20(1):15–27.
    External Resources
  26. Satomi E, Teodoro WR, Parra ER, Fernandes TD, Velosa AP, Capelozzi VL, et al. Changes in histoanatomical distribution of types I, III and V collagen promote adaptative remodeling in posterior tibial tendon rupture. Clinics. 2008 Feb;63(1):9–14.
    External Resources
  27. Godoy-Santos AL, Ranzoni L, Teodoro WR, Capelozzi V, Giglio P, Fernandes TD, et al. Increased cytokine levels and histological changes in cartilage, synovial cells and synovial fluid after malleolar fractures. Injury. 2017 Oct;48 Suppl 4:S27–33.
    External Resources
  28. Diniz-Fernandes T, Godoy-Santos AL, Santos MC, Pontin P, Pereira CA, Jardim YJ, et al. Matrix metalloproteinase-1 (MMP-1) and (MMP-8) gene polymorphisms promote increase and remodeling of the collagen III and V in posterior tibial tendinopathy. Histol Histopathol. 2018 Sep;33(9):929–36.
    External Resources
  29. Martins V, Teodoro WR, Velosa AP, Andrade P, Farhat C, Fabro AT, et al. Butylated hydroxytoluene induces type-V collagen and overexpression of remodeling genes/proteins in experimental lung fibrosis. Histol Histopathol. 2018 Oct;33(10):1111–23.
    External Resources
  30. Sant'Ana PG, Batah SS, Leao PS, Teodoro WR, de Souza SLB, Ferreira Mota GA, et al. Heart remodeling produced by aortic stenosis promotes cardiomyocyte apoptosis mediated by collagen V imbalance. Pathophysiology. 2018 Dec;25(4):373-79.
    External Resources
  31. Balancin ML, Teodoro WR, Farhat C, de Miranda TJ, Assato AK, de Souza Silva NA, et al. An integrative histopathologic clustering model based on immuno-matrix elements to predict the risk of death in malignant mesothelioma. Cancer Med. 2020 Jul;9(13):4836–49.
    External Resources
  32. Leite-Junior JH, Garcia CS, Souza-Fernandes AB, Silva PL, Ornellas DS, Larangeira AP, et al. Methylprednisolone improves lung mechanics and reduces the inflammatory response in pulmonary but not in extrapulmonary mild acute lung injury in mice. Crit Care Med. 2008 Sep;36(9):2621–8.
    External Resources
  33. de Oliveira MV, Rocha NN, Santos RS, Rocco MR, de Magalhães RF, Silva JD, et al. Endotoxin-induced emphysema exacerbation: a novel model of chronic obstructive pulmonary disease exacerbations causing cardiopulmonary impairment and diaphragm dysfunction. Front Physiol. 2019 May;10:664.
    External Resources
  34. Felix RG, Bovolato AL, Cotrim OS, Leao PD, Batah SS, Golim MA, et al. Adipose-derived stem cells and adipose-derived stem cell- conditioned medium modulate in situ imbalance between collagen I- and collagen V-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis. Histol Histopathol. 2020;35(3):289–301.
    External Resources
  35. Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS. 1988 May;96(5):379–94.
    External Resources
  36. Weibel ER. Lung morphometry: the link between structure and function. Cell Tissue Res. 2017 Mar;367(3):413–26.
    External Resources
  37. Meyrick B, Gamble W, Reid L. Development of Crotalaria pulmonary hypertension: hemodynamic and structural study. Am J Physiol. 1980 Nov;239(5):H692–702.
    External Resources
  38. Nogueira-Ferreira R, Vitorino R, Ferreira R, Henriques-Coelho T. Exploring the monocrotaline animal model for the study of pulmonary arterial hypertension: a network approach. Pulm Pharmacol Ther. 2015 Dec;35:8–16.
    External Resources
  39. Voelkel NF, Tuder RM. Hypoxia-induced pulmonary vascular remodeling: a model for what human disease? J Clin Invest. 2000 Sep;106(6):733–8.
    External Resources
  40. Gomez-Arroyo JG, Farkas L, Alhussaini AA, Farkas D, Kraskauskas D, Voelkel NF, et al. The monocrotaline model of pulmonary hypertension in perspective. Am J Physiol Lung Cell Mol Physiol. 2012 Feb;302(4):L363–9.
    External Resources
  41. Pietra GG, Capron F, Stewart S, Leone O, Humbert M, Robbins IM, et al. Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol. 2004 Jun;43(12 Suppl S):25S–32S.
    External Resources
  42. Jonigk D, Golpon H, Bockmeyer CL, Maegel L, Hoeper MM, Gottlieb J, et al. Plexiform lesions in pulmonary arterial hypertension composition, architecture, and microenvironment. Am J Pathol. 2011 Jul;179(1):167–79.
    External Resources
  43. Eyden BP. Brief review of the fibronexus and its significance for myofibroblastic differentiation and tumor diagnosis. Ultrastruct Pathol. 1993 Nov-Dec;17(6):611–22.
    External Resources
  44. Schermuly RT, Ghofrani HA, Wilkins MR, Grimminger F. Mechanisms of disease: pulmonary arterial hypertension. Nat Rev Cardiol. 2011 Jun;8(8):443–55.
    External Resources
  45. Fabro AT, da Silva PH, Zocolaro WS, de Almeida MS, Rangel MP, de Oliveira CC, et al. The Th17 pathway in the peripheral lung microenvironment interacts with expression of collagen V in the late state of experimental pulmonary fibrosis. Immunobiology. 2015 Jan;220(1):124–35.
    External Resources
  46. Iwanami K, Matsumoto I, Tanaka-Watanabe Y, Inoue A, Mihara M, Ohsugi Y, et al. Crucial role of the interleukin-6/interleukin-17 cytokine axis in the induction of arthritis by glucose-6-phosphate isomerase. Arthritis Rheum. 2008 Mar;58(3):754–63.
    External Resources
  47. Ricard N, Tu L, Le Hiress M, Huertas A, Phan C, Thuillet R, et al. Increased pericyte coverage mediated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth muscle-like cells in pulmonary hypertension. Circulation. 2014 Apr;129(15):1586–97.
    External Resources
  48. Maarman G, Lecour S, Butrous G, Thienemann F, Sliwa K. A comprehensive review: the evolution of animal models in pulmonary hypertension research; are we there yet? Pulm Circ. 2013 Dec;3(4):739–56.
    External Resources
  49. Arcot SS, Lipke DW, Gillespie MN, Olson JW. Alterations of growth factor transcripts in rat lungs during development of monocrotaline-induced pulmonary hypertension. Biochem Pharmacol. 1993 Sep;46(6):1086–91.
    External Resources
  50. Morty RE, Nejman B, Kwapiszewska G, Hecker M, Zakrzewicz A, Kouri FM, et al. Dysregulated bone morphogenetic protein signaling in monocrotaline-induced pulmonary arterial hypertension. Arterioscler Thromb Vasc Biol. 2007 May;27(5):1072–8.
    External Resources
  51. Alencar AK, Pereira SL, Montagnoli TL, Maia RC, Kümmerle AE, Landgraf SS, et al. Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats. Br J Pharmacol. 2013 Jul;169(5):953–62.
    External Resources
  52. Dart ML, Jankowska-Gan E, Huang G, Roenneburg DA, Keller MR, Torrealba JR, et al. Interleukin-17-dependent autoimmunity to collagen type V in atherosclerosis. Circ Res. 2010 Oct;107(9):1106–16.
    External Resources
  53. Pang L, Qi J, Gao Y, Jin H, Du J. Adrenomedullin alleviates pulmonary artery collagen accumulation in rats with pulmonary hypertension induced by high blood flow. Peptides. 2014 Apr;54:101–7.
    External Resources

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