In vivo knockdown of intersectin-1s alters endothelial cell phenotype and causes microvascular remodeling in the mouse lungs
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Intersectin-1s (ITSN-1s) is a general endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, via MEK/Erk1/2MAPK signaling. To investigate the in vivo effects of ITSN-1s deficiency and the resulting ECs apoptosis on pulmonary vasculature and lung homeostasis, we used an ITSN-1s knocked-down (KDITSN) mouse generated by repeated delivery of a specific siRNA targeting ITSN-1 gene (siRNAITSN). Biochemical and histological analyses as well as electron microscopy (EM) revealed that acute KDITSN [3-days (3d) post-siRNAITSN treatment] inhibited Erk1/2MAPK pro-survival signaling, causing significant ECs apoptosis and lung injury; at 10d of KDITSN, caspase-3 activation was at peak, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive ECs showed 3.4-fold increase, the mean linear intercept (MLI) showed 48 % augment and pulmonary microvessel density as revealed by aquaporin-1 staining (AQP-1) decreased by 30 %, all compared to controls; pulmonary function was altered. Concomitantly, expression of several growth factors known to activate Erk1/2MAPK and suppress Bad pro-apoptotic activity increased. KDITSN altered Smads activity, downstream of the transforming growth factor beta-receptor-1 (TβR1), as shown by subcellular fractionation and immunoblot analyses. Moreover, 24d post-siRNAITSN, surviving ECs became hyper-proliferative and apoptotic-resistant against ITSN-1s deficiency, as demonstrated by EM imaging, 5-bromo-deoxyuridine (BrdU) incorporation and Bad-Ser112/155 phosphorylation, respectively, leading to increased microvessel density and repair of the injured lungs, as well as matrix deposition. In sum, ECs endocytic dysfunction and apoptotic death caused by KDITSN contribute to the initial lung injury and microvascular loss, followed by endothelial phenotypic changes and microvascular remodeling in the remaining murine pulmonary microvascular bed.
KeywordsApoptosis Endothelial cells Microvascular remodeling Phenotypic changes siRNA
Intersectin-1s (ITSN-1s) is a member of ITSN family of proteins, best-known for its critical role in coordinating distinct steps in clathrin-coated vesicles (CCVs) and caveolae endocytosis, [1, 2, 3, 4]. Due to its endocytic function and multimodular structure, ITSN-1s links growth factors receptors and regulatory proteins predominantly localized in CCVs and caveolae, to signaling networks [1, 2, 5, 6]. ITSN-1s activates Ras, via its association with mSOS, a GEF for Ras, leading to downstream Erk1/2MAPK signaling activity . Previous studies indicated that downregulation of ITSN-1s via siRNA inhibits Erk1/2MAPK and its direct activator MEK, leading to mitochondrial apoptosis of cultured human lung microvascular ECs . The findings are consistent with pro-survival signaling orchestrated by ITSN-1s via mSOS/Ras/Erk1/2MAPK pathway, known to play a pivotal role in cell survival, proliferation and vascular remodeling, [9, 10, 11].
ECs dysfunction and apoptotic death are common events in many pathological conditions such as sepsis, inflammatory syndromes, atherosclerosis, and pulmonary arterial hypertension (PAH), . Recent studies showed that increased pulmonary ECs apoptosis caused by loss-of-function mutations in bone morphogenetic protein receptor-2 (BMPR2) may represent a possible initiating mechanism in PAH, . Moreover, studies using chronically hypoxic SU5416-treated rats indicated that increased apoptosis of ECs created conditions favoring the emergence of apoptosis-resistant cells and that blockade of ECs growth factors receptors worsen the pathological vascular remodeling .
ITSN-1s is one of the recently identified substrates for the cytotoxic protease granzyme B (GrB), , raising the possibility that during inflammatory processes, major pathogenic components of pulmonary vascular remodeling, , ITSN-1s can be cleaved and thus, expression of full-length protein reduced. Recently, we have shown that ITSN-1s deficiency is relevant to the pro-inflammatory ECs dysfunction induced by lipopolysaccharide; the decrease in ITSN-1s mRNA and protein expression were countered by Bcl-XL and survivin upregulation, as well as Bim downregulation, events thought to protect ECs from impending apoptosis . Little is known about ITSN-1s deficiency and its implications in human lung diseases, since no studies have examined the consequences of KDITSN and the subsequent ECs dysfunction and apoptotic death in vivo. With the recent introduction of siRNA, the silencing of genes involved in pathologic mechanisms is carried out extensively in vitro, but more limited in vivo, due to either inefficient KD or adverse effects, such as immune responses, toxicity or off-target effects . Recently, using cationic liposomes/siRNAITSN complexes delivered retro-orbitally into mice we knocked down ITSN-1s protein and mRNA levels in mouse lungs for 24 consecutive days . The amounts of siRNAITSN injected (100 μg siRNA/mouse) were significantly lower compared to previously reported data , and sufficient for efficacy with no detectable adverse effects. Using this KDITSN mouse model, we have shown that lung ECs deficient of ITSN-1s, while impaired in caveolae and CCVs endocytosis, show up-regulation of alternative endocytic pathways (i.e. enlarged endocytic structures, membranous rings and tubules, etc.,) to compensate for deficient vesicular trafficking . It is well-known that endocytosis and vesicular trafficking have significant impact on growth factors receptors signaling/activity and their associated proteins and downstream targets [21, 22, 23]. Endocytic dysfunction and altered intracellular trafficking and signaling of cell surface receptors such as TβRs, BMPR2 and tyrosine kinase receptors [i.e. vascular endothelial growth factor receptor (VEGF-R), platelet-derived growth factor receptor, (PDGF-R), insulin receptor (IR) etc.] have been implicated in the pathogenesis of PAH, emphysema and chronic obstructive pulmonary disease [24, 25, 26, 27], pointing to implications of disrupted intracellular membrane trafficking in the pathobiology of vascular remodeling. However, studies on the potential involvement of ECs dysfunctional endocytic membrane traffic and the consequences on growth factor(s) receptors signaling in the pathology of lung microvascular remodeling are limited. Thus, the aim of this study was to elucidate the in vivo effects of ECs apoptosis and impaired endocytic membrane trafficking caused by ITSN-1s deficiency on mouse lung vasculature and lung homeostasis, using the lung-specific KDITSN mouse model we generated . Here, we show that specific downregulation of ITSN-1s expression in mouse lungs causes ECs death and lung injury, followed by endothelial phenotypic changes toward hyper-proliferation and apoptosis-resistance, leading to lung repair and microvascular remodeling in the remaining pulmonary microvascular bed. Overall these findings established an important role of ITSN-1s in the ECs function and maintenance of lung homeostasis, while illustrating the effectiveness of siRNA in vivo.
Materials and methods
Delivery of siRNAITSN to mouse lungs
Young mice (6–8 weeks), as well older mice (24 weeks) were used in the studies. A specific ITSN-1s siRNA sequence (100 μg siRNA/mouse) targeting ITSN-1 gene was delivered via cationic liposomes, by retro-orbital injection, into mouse lungs as in [19, 28]. Pilot studies we performed to determine the siRNAITSN amount needed for efficient KDITSN without structural alteration of mouse organs, lung included. Chronic inhibition of ITSN-1s, was achieved by repeated siRNAITSN/liposomes delivery, every 72 h, for 24d as in . Mice were sacrificed at 3, 10 15 and 24d; Three to four mice per experimental condition [controls (wt-mice, vehicle- and non-specific siRNA-treated mice) and siRNAITSN-treated mice] were used; all these experiments were repeated three times. No mouse mortality was recorded during the 24d of this experimental approach. All animal studies were performed in accordance with the guidelines of the University Institutional Animal Care and Use Committee. All surgeries were performed under anesthesia using i.p. delivery of 1 ml/kg body weight of ketamine hydrochloride/xylazine hydrochloride solution, (Sigma, St. Louis, MO).
Lungs free of blood by in situ perfusion (1.5 ml/min warm Hank’s solution) were homogenized in 150 mM NaCl, 50 mM Tris, pH 8.0 and protease inhibitors; total lysates were prepared by adding NP-40, to a final concentration of 1.0 %, for 2 h, at 4 °C. The ensuing lysates were clarified by centrifugation in a Beckman ultracentrifuge with a TLA-55 rotor, at 45,000 rpm, for 45 min, at 4 °C. Protein concentration was determined by the microBCA, (Pierce Biotechnology, Rockford, IL), with a bovine serum albumin (BSA) standard.
Nuclear and cytosolic fractions of mouse lungs were prepared as in . Lung tissue, free of blood by in situ perfusion of Hank’s solution, was washed twice with PBS and then transferred to hypotonic buffer (250 mM sucrose, 50 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 1 mM DTT, and 1 mM phenylmethyl sulfonyl fluoride) for 30 min to allow swelling. The tissue was then Dounce-homogenized (20–30 strokes) with a tight-fitting pestle. The homogenate was centrifuged in a refrigerated Beckman benchtop centrifuge at 800×g for 15 min; the postnuclear supernatant served as cytosol, while the pellet, which contains the nuclei, was resuspended in a lysis buffer and centrifuged again as above. The supernatant served as the “nuclear” fraction and further used for assessing Smads nuclear translocation.
Western blot and densitometry
Total protein (70 μg/lane) of mouse lung lysates was subjected to 4–20 % SDS PAGE, transferred to nitrocellulose membranes, followed by Western blot as in . The antibodies (Abs)—AQP-1, Smad4/7 pAbs, histone-3, VEGF-A, a dual BMP-2/4 mAbs and phospho-Smad2 pAb, (Santa Cruz Biotechnology, Santa Cruz, CA), Bad and phospho-Bad pAbs, BrdU mAb, and phospho-Erk1/2 mAb, Erk1/2, TGFβ Smad1, phospho-Smad1/5/8, caspase-3 pAbs (Cell Signaling, Danvers, MA) phospho-Smad3 pAb (Abcam, Cambridge, MA) were used as recommended by the manufacturers, at dilutions established in preliminary experiments. ITSN-1s mAb (BD Biosciences, San Jose, CA) was used throughout the study to evaluate the efficient KDITSN in mouse lung tissue. The bands of immunoreactivity were visualized using appropriate horseradish peroxidase-conjugated Abs (eBioscience, San Diego, CA) and enhanced chemiluminescent substrate (Pierce, Rockford, IL). HyBlot CL films were subjected to densitometric analysis using ImageJ1.37v software.
Controls and siRNAITSN-treated mouse lungs were inflated with 1 % low-melting point agarose in 10 % formalin at a constant pressure of 25 cm H2O, allowing for homogenous expansion of lung parenchyma, and then fixed in 10 % paraformaldehyde for 48 h and paraffin-embedded [31, 32]. Thin sections (6 μm), were stained with hematoxylin/eosin (H&E) for lung tissue histological evaluation. Images (5 per lung section, 3 sections per mouse, 3–4 mice per group) were acquired with a 10 × lens and used for morphological assessment. MLI, a measure of interalveolar wall distance was evaluated on micrographs (35 micrographs randomly selected/specimen/3 different experiments) as in . Briefly, the length of a line drawn across the lung section was divided by the total number of intercepts encountered in 100 lines per each section analyzed.
Measurement of lung mechanics
Assessment of pulmonary lung function in controls and liposome/siRNAITSN-treated mice was performed using the Flexivent system (Scireq, Montreal, Canada) as in [33, 34]. Briefly, mice were anesthetized with inhaled isoflurane in oxygen and orotracheally intubated with a 20-gauge intravenous cannula under direct vision. Isoflurane anesthesia was maintained throughout the measurements, and the mice were hyperventilated to eliminate spontaneous ventilation. Before testing each mouse, tube calibration was performed to remove the mechanical impedance of the tracheal cannula. Then, starting at functional residual capacity, the Flexivent was programmed to deliver seven inspiratory volume steps, for a total volume of 1 ml, followed by seven expiratory steps, pausing at each step for at least 1 s. Using this approach we evaluated the lung compliance (CL) and resistance (RL) as well as the inspiratory capacity (IC). Statistical analysis was performed with SigmaStat software using one-way ANOVA testing method (n = 4/each time point investigated). Statistical difference was accepted at P < 0.05. All data are presented as mean ± SEM.
Lectin staining was performed using biotinylated Griffonia Symplicifolia-1 (GS-1; Sigma) which binds specifically to α-galactosyl residues from microvascular endothelium . After dewaxing and rehydration in PBS, the sections were blocked for unspecific binding with 1 % BSA in PBS, for 30 min. GS-1 (50 μg/ml) was applied for 1 h, at RT, in a humidified chamber. After washing, sections were incubated with NeutrAvidin dyelight (Sigma) for 30 min, and mounted with Prolong Antifade reagent (Molecular Probes).
Aquaporin-1 (AQP-1) staining and morphometry
Deparaffinized and hydrated tissue sections were incubated for 15 min with 1.5 % H2O2 for blocking the endogenous peroxidase activity, followed by 1 h in 10 mM sodium citrate, pH 6.0, at 65 °C for antigen unmasking, as in . Incubation with AQP-1 Ab for 30 min, at RT was followed by biotinylated universal Ab diluted in 2 % mouse serum and Vectastain ABC reagent (Vector Labs.) for 30 min, each. NovaRed substrate (Vector Labs.) was used for developing the red color. The number of microvessels labeled by AQP-1 was estimated by superimposing a rectangular grid over the sections and the microvessels included within the boundaries of each rectangle, were counted. The criterion used for identifying and counting microvessels was a circular/elliptical contour (<9 μm diameter) labeled by the AQP-1 Ab. When a capillary placed on the grid lines, it was considered only one time. Images (5 per lung section, 3 sections per mouse, and 3–4 mice per group) were acquired with a 63 × lens and used for morphological assessment. A minimum of 25 randomly chosen power fields per section form controls and ITSN-1s deficient mice were measured at a final magnification of 300×.
Apoptosis and cell proliferation assays
TUNEL was carried out as per manufacturer’s instructions, (Roche, Indianapolis, IN). Paraffin-embedded sections from controls and siRNAITSN-treated mouse lungs were deparaffinized, washed in PBS and treated with proteinase K, for 20 min at RT. The TUNEL mixture was applied for 60 min in a humidified atmosphere at 37 °C, in the dark. After washing, the sections were mounted using Prolong Antifade reagent. Lung sections incubated with TUNEL solution in the absence of TUNEL enzyme solution were used as negative control. TUNEL-positive ECs were identified by fluorescence microscopy using an excitation wavelength of 488 nm. Lung autofluorescence was reduced by incubation of lung sections, for 5 min with sulphorhodamin solution.
Mouse lungs ECs were labeled with BrdU solution (Roche, Indianapolis, IN) by intraperitoneal injection every 24 h, for two consecutive days. Paraffin-embedded sections were dewaxed and rehydrated in PBS for 10 min. Trypsin solution was applied for 5 min. The sections were denatured in 4 M HCl for 15 min and washed again in PBS. Next, each preparation was incubated with anti-BrdU/FITC Ab, for 45 min, at 37 °C in a humid chamber. After the last washing in PBS, the slides were mounted as above.
Quantification of BrdU- and TUNEL-positive nuclei was performed on medium-sized (50 ≥ diameter ≤ 100 μm) blood vessels. Using Zeiss Axiovision software we measured the diameter of the vessels by drawing an imaginary line perpendicular to the direction of the largest profile dimension which was then averaged with the value at its greatest width. A minimum of 25 vessels per section (3 sections per mouse, 3–4 mice per group) were used for counting the number of TUNEL- and BrdU-positive ECs nuclei. Data was normalized per 25 vessels.
BrdU-positive ECs were counted on 25 alveolar profiles in controls and siRNAITSN-treated mouse lung sections. For treated specimens, only alveolar profiles with more than one ECs TUNEL-positive were considered on randomly chosen middle power fields.
Paraffin-embedded mouse lung sections were deparaffinized and hydrated in distilled water, stained in hematoxilyn and then subjected to Picrosirius Red (Polysciences) staining as instructed by the manufacturer. Finally, sections were dehydrated, cleared in xylene, and mounted with Paramount (Fischer Scientific). Picrosirius Red staining was imaged using a Zeiss Axio Observer Z1 Motorized Inverted Microscope; morphometry was performed using ImageJ1.37v software. Areas of interest were defined using 20–25 high power microscopic fields/mouse/experimental condition. The per area density of binding/staining was obtained by averaging the percentage values obtained for each area of interest analyzed.
Statistical analysis was performed using Student’s two-tailed, unpaired t test for comparison between groups, for collagen staining and ANOVA testing for all other lung morphometry. Data are reported as mean ± SEM, Statistical significance was accepted at P < 0.05.
Mouse lungs, free of blood, were fixed by perfusion with 3 % paraformaldehyde and 1.5 % glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.3 . Excised tissue was fixed in the same fixative mixture for one additional hour, at RT, post-fixed in Palade’s 1 % OsO4 on ice, and then stained with Kellenberger uranyl acetate. Selected specimens were dehydrated in graded ethanol and embedded in Epon812. Thin sections cut from blocks were mounted on nickel grids, stained with 2 % uranyl acetate and lead citrate, and finally examined and micrographed in a Joel 1220 TEM.
Specific KDITSN in mouse lungs
ITSN-1s protein and mRNA levels were monitored at several time points after siRNAITSN delivery by Western blot and RT- and qPCR as in . ITSN-1s protein and mRNA levels in siRNAITSN-treated mouse lungs were about 75 % lower by reference to controls. Delivery of empty-liposomes or liposomes containing the non-specific siRNA did not affect the levels of ITSN-1s protein or mRNA, compared to controls. Comparable ITSN-1s downregulation, with the same timeline as in the lung, was detected in the brain, while in the heart, kidneys and liver was less efficient (not shown). This KDITSN murine model generated by liposome delivery of the siRNAITSN was used to further confirm in vivo that ITSN-1s deficiency induces ECs apoptosis in the pulmonary vasculature while affecting lung homeostasis.
Acute KDITSN causes ECs apoptosis, microvessel loss and mouse lung injury
Morphometric analysis of ECs apoptosis, proliferation and microvessel density in the lungs of KDITSN mouse. The number of lung microvessels labeled by AQP-1 and GS-1 lectin was normalized per 30,000 μm2 surface area
TUNEL + ECs/25 medium-sized vessels
48.0 ± 2.4
115 ± 2.9
165 ± 2.8
25 ± 2.0
AQP-1 microvessels, Ø < 9 μm
38.8 ± 4.0
28.81 ± 2.4
25.6 ± 4.0
BrdU + ECs/25 medium-sized vessels
83.0 ± 5.0
154 ± 3.8
173 ± 5.6
BrdU (1.5 × 102 μm alveolar wall length)
4.4 ± 1.0
8.3 ± 1.5
9.4 ± 1.8
GS-1 lectin(microvessels, Ø < 20 μm)
365 ± 9.0
270 ± 4.5
443 ± 7.1
Key morphological features of ECs apoptosis and lung injury induced by KDITSN were also identified by detailed EM surveys of the mouse lungs deficient of ITSN-1s. Frequently, ECs with increased number of mitochondrial units, many of them with abnormal morphology, Golgi apparatus with swollen and unstacked cisternae (Fig. 3C, c3), as well as ECs nuclei displaying evident signs of chromatin condensation (Fig. 3C, c4), were noticed in KDITSN mouse specimens. For comparison, Fig. 3C, c1, c2 illustrate fragments of wild-type mouse lungs ECs with normal mitochondria, well-organized Golgi, as well as a healthy nucleus. Caveolae, the main vesicular carriers of ECs are numerous and frequently found open to the lumen or apparently free in the cytosol (Fig. 3C, c.1.1). We have often observed in the lungs of KDITSN mice ECs displaying finger-like protrusions in the lumen of the blood vessels (Fig. 3C, c5, arrowheads), evidence of a dysfunctional activated EC. Recent studies indicate that the membraneous protrusions are very dynamic structures, likely involved in the release of circulating membrane particles, involved in the maintenance and preservation of cellular homeostasis and in promoting defense mechanisms [41, 42]. In addition some of these protrusions are physiologically relevant since they may function in pinocytosis to compensate for deficient endocytic membrane traffic via caveolae and CCVs . Significantly, as a consequence of endothelial injury caused by ITSN-1s deficiency, the permeability of the alveolo-capillary unit was affected, leakage of the protein-rich fluid from the vascular to the interstitial space occurred, causing dilation of the interstitial space at the level of the alveolar septa (Fig. 3C, c4, c5). Overall, we noticed that the lung injury caused by KDITSN was patchy, mainly localized at the level of the alveolo-capillary units, centered on small vessels (capillaries, post-capillary venules) and around some mid-sized vessels (collecting and muscular venules). Most likely, this patchy distribution reflects the efficient delivery of the siRNAITSN and thus, downregulation of ITSN-1s. Altogether, the observations indicated that ITSN-1s expression is important for ECs function and survival and that ECs death is an important feature of lung injury in this mouse model.
Time course of ECs apoptosis in mouse lungs with chronic KDITSN
Densitometric analysis indicated more than 3-fold increase in cleaved caspase-3 at 10d, compared to untreated mice (Fig. 4B). TUNEL (Fig. 4C, c1), followed by CD31 Ab staining for specific detection of ECs within the alveolar wall, (Fig. 4C, c2) also revealed increased number of apoptotic ECs nuclei at 10d (Fig. 4C, c3, arrows) compared to 3d KDITSN. We have also detected TUNEL-positive alveolar epithelial cells (Fig. 4C, c3, open arrows). Their apoptotic death can be due to both, (i) the excessive death of ECs and subsequent collapse of the capillary bed, essential for the maintenance of alveolar septa and (ii) a direct effect of KDITSN at this level. When the extent of ECs apoptosis in medium-sized vessels was evaluated by TUNEL, morphometric analysis indicated that the number of apoptotic ECs reached the highest values at 10d of KDITSN, (Fig. 4D, Table 1), consistent with caspases-3 activation. Moreover, AQP-1 Ab staining and morphometric analysis indicated a further loss of pulmonary microvessels; at 10d, the mean number of lung microvessels per examined area reached the lowest value of 25.6 ± 4, (Table 1). Even with significant apoptosis, no mortality occurred among mice with KDITSN for 24 consecutive days. Despite continuous and efficient KDITSN, at 24d, the number of apoptotic ECs in medium-sized vessels decreased by 50 % compared to controls and by 85 % compared to KDITSN, at 10d (Table 1), while in the alveolar wall they were detected only occasionally. All together, these observations suggested that extensive ECs death, recorded at 10d of KDITSN, may stimulate ECs hyper-proliferation and perhaps, apoptosis-resistance.
Chronic KDITSN mouse lungs show phenotypically-altered ECs
The increased cell proliferation in chronic KDITSN strongly indicated that the survival signaling was restored. Therefore, we evaluated the Erk1/2MAPK activation, known to be affected by ITSN-1s deficiency . Erk1/2MAPK phosphorylation was barely detected at 72 h post-siRNAITSN delivery (Fig. 5B); however, by 10d, Erk1/2 activation increased about 50 % over the baseline (Fig. 5B, line a), reached a peak at 15d and was still above controls, at 24d of KDITSN; no changes in total Erk1/2MAPK were detected. Control experiments using mice injected with siRNActrl/liposomes complexes (Fig. 5B, line b) and liposomes only (Fig. 5B, line c), 72 h post injection, indicated no significant changes in Erk1/2 phosphorylation compared to the baseline (Fig. 5B, line a). Since Erk1/2MAPK activation can be related to increased expression of growth factors , and since the Ras/Erk1/2MAPK is a major signaling pathway downstream of TβR1 , we next evaluated the expression of TGF a multi-functional cytokine shown to be involved in ECs proliferation, survival and maintenance of vascular integrity . A modest boost of mature TGFβ at 72–20 % increase compared to control, and a further a 3-fold increase at 10d post-siRNAITSN treatment, when apoptosis was at peak (Fig. 5C, D), suggested that TGFβ may account for Erk1/2MAPK activation in KDITSN mice. A gradual decrease to under baseline levels at 24d, were consistent with the stimulatory role of low doses of TGFβ on ECs proliferation . Significant immunoreactivity for the TGFβ homodimeric pro-protein (Mr 74-kDa), was detected at all time points, (Fig. 5C). A dual BMP-2/4 Ab, revealed that the level of mature BMP-2/4, two TGFβ family members able to initiate ECs proliferation [47, 48, 49], was increased by 2.2-fold at 15d and 1.7-fold at 24d KDITSN, compared to controls (Fig. 5C, D). Finally, VEGF-A, a multifunctional cytokine that stimulates ECs to survive, proliferate and alter the pattern of their gene expression , and whose downstream signaling includes Erk1/2MAPK activation, paralleled BMP-2/4 (Fig. 5C, D). One of likely targets of phosphorylation, by growth factors-activated Erk1/2MAPK is the Bcl-2 family Bad [51, 52]. Bad phosphorylation causes inactivation of its pro-apoptotic properties by blocking the interaction with Bcl-XL, [44, 51]. Immunoblotting using site-specific phospho Abs indicated Bad phosphorylation at, Ser112 and Ser155, (Fig. 5E); No phosphorylation of Bad-Ser136, a preferred substrate for PI3K/Akt , was identified. PI3K/Akt activation, a downstream target of VEGF signaling , was not detected in this cellular context (not shown). We concluded that these multi-functional growth factors may act synergistically to re-establish pro-survival signaling and engage anti-apoptotic pathways, shifting the ECs phenotype toward hyper-proliferation and apoptosis-resistance against ITSN-1s deficiency.
Chronic KDITSN induces alveolar and vascular remodeling
Lung cells apoptotic death altered lung mechanics
CL, RL and IC in KDITSN mice were evaluated with Flexivent at 72 h, 10 and 24d after siRNAITSN treatment. As expected, based on extensive ECs apoptosis and pulmonary edema recorded at 72 h post-siRNAITSN treatment, CL was decreased by comparison to wild-type mice, when the mean values where compared. (Fig. 6C, c1); IC was not significantly modified (Fig. 6C, c2). At the same time point we found increase in RL (Fig. 6C, c3), most likely due to increased pulmonary recoil, an expected effect of lung fluid accumulation. Interestingly however, at 10d post-siRNAITSN treatment, the pulmonary function of KDITSN mice evolved toward increased CL, increased IC and low airway RL, compared to the wild-type or 72 h post-siRNA mouse lungs. This observation is consistent with the significant and persistent alveolar destruction, enlargement of the air space and increased MLI values. Given the ongoing compensatory cell proliferation, however, the state of endothelium ameliorates and the vascular leakage leading to accumulation of fluid within the interstitial space decreases, as already documented by us , fact that may account for CL values at this time point. In keeping with this observation, 24d post-siRNAITSN delivery, pulmonary function of KDITSN mice show no notable differences compared to controls.
KDITSN mice present increased pulmonary microvessel density and altered Smad activity
Downstream of TGFβ and BMP-2/4 signaling are Smad proteins, involved in ECs proliferation and angiogenesis . First, Smad1/5/8 activity was evaluated by Western blot analyses of both cytosol and nuclear fractions prepared from lung tissue of control and KDITSN mice at 3, 10, 15 and 24d post-siRNAITSN delivery, using a phospho-Smad1/5/8 Ab, (Fig. 7C). Phosphorylation of Smad1/5/8/, downstream events of both ALK1, a TβR1 whose expression is restricted to ECs, and BMPR2  was slightly increased in both fractions at 72 h post-siRNAITSN treatment; then a gradual decrease, about 30 % lower compared to controls and 72 h post-siRNAITSN, was detected. Since previous reports demonstrated that TβR1 signaling is not directly affected by impaired endocytosis , and that inhibition of BMPR2 endocytosis does not affect Smad1/5/8 phosphorylation, but affects their nuclear translocation , it is likely that in a cellular environment deficient in caveolae and CCVs endocytosis, the BMPR2 endocytic traffic is altered due to upregulation of compensatory alternative endocytic pathways, . Both Smad1/5/8 phosphorylation and nuclear translocation still occur, but less efficiently compared to controls. At 72 h post-siRNAITSN, the alternative endocytic pathways do not function efficiently enough (Predescu, 2012) to affect BMPR2 endocytic internalization; the receptor is still on cell surface and less internalized due to caveolae and CCVs impaired endocytosis, and thus its signaling may be enhanced, resulting in increased Smad1/5/8 phosphorylation and nuclear translocation, at this time point.
Moreover, the Smad2/3, downstream of TβR1 show decreased levels of phosphorylation, Fig. 7D. The levels of Smad2/3 are unchanged, suggesting that Smad2 and Smad3 are not targeted for degradation. It appears that the typical TβR1/ALK5 signaling has been shifted from the Smad2/3 activation toward a less common Ras/Erk1/2MAPK pathway, with protective effects on ECs and lung vasculature. Significantly, as already reported, Smad2/3 inhibition is consistent with the ECs proliferation .
To rule out a possible involvement of Smad 4 and inhibitory Smad 7 in the inhibition of Smad2/3 activity, we analyzed by Western blot the expression of the two Smad proteins in mouse lung lysates (Online Resource 1). No changes in the expression of the common Smad 4 were detected. The levels of the inhibitory Smad7, display a gradual decrease, reaching a 30 % decrease at 24d post-siRNAITSN delivery compared to Smad7 in the lung lysates of wt-mice. The observations further support the concept that the impaired endocytic traffic of TβR1 and activation of Erk1/2MAPK account for Smad2/3 inhibition.
In this study we evaluated the in vivo effects of ITSN-1s deficiency on pulmonary ECs apoptosis and lung homeostasis using a KDITSN mouse model in which ITSN-1s was efficiently and specifically inhibited for 24 consecutive days, via i.v. delivery of siRNAITSN/cationic liposomes. Acute KDITSN and loss of Erk1/2MAPK survival signaling caused significant ECs apoptosis leading to endothelial damage, microvessels loss and alveolar destruction. These results are in keeping with our previous studies showing that ITSN-1s deficiency interferes with ECs function and survival by inhibition of MEK/Erk1/2 phosphorylation . Increased ECs death as revealed by TUNEL and caspase-3 activation began at 3d and peaked at 10d of KDITSN. As a consequence, lung injury occurred rapidly, allowing us for a detailed time course analysis of structural destruction and disease progression.
Studies have shown that in the lungs of chronically hypoxic rats treated with the VEGFR-2 inhibitor SU5416, pulmonary ECs death preceded development of severe PAH, associated with precapillary arterial occlusion by proliferating ECs . Mouse lung ECs death, as caused by KDITSN, was a prerequisite for the following ECs proliferation, repair and remodeling process. Within days, these normally quiescent cells with a low turnover rate, began to hyper-proliferate, indicating that the survival signaling, lost due to KDITSN, was re-established.
Sustained activation of Erk1/2 is necessary for cells progressing from G1 to S-phase and it is usually associated with induction of D-type cyclins and assembly with their catalytic partners that phosphorylate the retinoblastoma proteins resulting in release of E2F and transcription of genes required for DNA replication . Thus, Erk1/2-Smad2/3 signaling switch may affect two well-known molecular mechanisms of cell growth arrest by TGFβ in the G1-phase of cell cycle, (i) downregulation of D type cyclins and cyclin-dependent kinases expression [62, 63, 64] and (ii) activation of cyclin-dependent kinases inhibitors , events that may account for initiation of the ECs hyper-proliferative phenotype.
Bad phosphorylation by growth factors activated Erk1/2MAPK, and inactivation of its pro-apoptotic properties, already demonstrated in cultured neurons and in models of cerebral ischemia,  may be part of the molecular mechanism leading to the EC apoptotic-resistant phenotype caused by chronic KDITSN and lung repair.
Although the crosstalk between the TGFβ and other signaling pathways was not addressed in detail in these experimental settings, the studies described here demonstrate that Erk1/2MAPK activation, Bad phosphorylation and the perturbed Smad signaling downstream of ALK5 and BMPR2, play an important role in lung ECs survival and injury repair.
Vascular remodeling induced by chronic KDITSN was assessed by a detailed survey of AQP-1 and GS-1 lectin labeling, widely used to visualize the mouse lung microvessels . Excessive apoptotic ECs death recorded at 72 h and 10d post-siRNA treatment, and the decrease of microvessels density were followed by ECs proliferation and microvascular repair/development process that not only restored the number of lung microvessels, but apparently, added new microvessels to the system. Increased expression of both VEGF-A, a central growth and survival factor for the ECs  and BMP2/4 already implicated in capillary sprouting via Erk1/2MAPK signaling , may be, at least partly responsible for this KDITSN mouse phenotype. Moreover, the apoptotic/proliferative ECs coexistence (high cellular death/growth) in the murine lung endothelium at 10d of KDITSN indicates the conversion time of normal, surviving ECs to a hyper-proliferative phenotype. What initiated out of necessity for tissue replenishment, progresses to microvascular remodeling due to a newly acquired hyper-proliferative ECs phenotype. As shown KDITSN in the mouse lungs also affected lung epithelial cells—either extensive ECs apoptosis or a direct siRNA effect on lung epithelium may account for the observed epithelial cells apoptotic death. Lung parenchymal proliferation might be expected as a result of increased number of blood vessels, providing the energy substrate for accelerated alveolar replenishment . Thus, it is not surprising that BrdU staining of lungs sections with chronic KDITSN revealed also a multitude of proliferative epithelial cells. At the end of the study no significant difference was found between MLI of these specimens and controls. Even if the MLI shows overall equality, lung morphology after 24d of ITSN-1s suppression did not resemble controls. Few increased airspace regions were in complementary distribution to various areas with closely packed alveoli, smaller in size than in controls. This data suggest that hyper-proliferative pulmonary ECs prompted pneumocytes proliferation, reactive to the favorable milieu created by the vascular development. While the role of epithelial cell apoptosis in lung repair and remodeling cannot be disregarded, given the efficient and specific delivery of siRNAITSN/cationic liposomes to pulmonary ECs and the high endothelial content of the lung tissue , the findings reported here are highly relevant for ECs of the lung.
During the 24d of ITSN-1s deficiency, pulmonary function of KDITSN mice evolved from low CL and increased RL at 72 h post-siRNA delivery, toward increased CL and decreased RL at 10d, while, at the end of the study both parameters were close to control values. Compensatory proliferation, observed at 10d post-siRNA treatment, contributed to the amelioration of endothelial injury and reduced the pulmonary fluid buildup, events that are behind the change in pulmonary mechanics at 10d. Depositions of matrix components (collagen and elastin fibers) and increase in interstitial cell population were commonly seen at the ultrastructural level in mouse lung vessels with chronic KDITSN. The remodeling of the alveolo-capillary functional unit (i.e. epithelial cells proliferation, collagen deposition within alveolar septum, increased microvessels density) further improved the CL, RL and IC of the injured lungs.
Taken together, our data showed that in the pulmonary tissues of mice deficient of ITSN-1s, impaired endocytic membrane traffic and initial ECs apoptosis was followed by ECs phenotypic changes toward hyper-proliferation and apoptosis-resistance against ITSN-1s deficiency as well as microvascular remodeling in the remaining pulmonary microvascular bed. Interestingly, TβR1 downstream signaling was apparently switched from the canonical Smad2/Smad3 toward Erk1/2MAPK. This signaling switch proved to be central for ECs survival, maintaining vascular integrity and lung homeostasis.
The findings are relevant considering that ITSN-1s is a general endocytic protein, a regulator of mitochondrial apoptosis and a GrB substrate . Under inflammatory conditions and increased GrB levels, loss of full-length ITSN-1s may cause endocytic dysfunction, loss of pro-survival signaling and apoptotic ECs death. As result, loss of blood vessels, damage of the alveolar wall, abnormal or incomplete tissue repair may occur. These events are major determinants able to cause during disease progression the selection of hyper-proliferative and apoptotic-resistant cell phenotypes . The findings implicate ITSN-1s, a key endocytic and pro-survival protein of lung endothelium, and the biological processes regulated by it (i.e., endocytosis, transcytosis, cell survival and apoptosis) in vascular remodeling and pathology of lung disorders.
We thank Nicki Watson (Whitehead Institute, Cambridge, MA) for technical support with the EM studies and Andy Hall (University of Illinois at Chicago, IL) for helping with CD31/BrdU and CD31/TUNEL immunostaining. This work was supported by National Institute of Health Grants R01HL089462, R01HL089462-02S1 and American Heart Association SDG0635175N (to S.P.).
Conflict of interest
The authors have no conflicts of interest to disclose.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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