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Am J Physiol Lung Cell Mol Physiol 291: L1159-L1168, 2006. First published August 4, 2006; doi:10.1152/ajplung.00168.2006
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Hypoxia-induced mitogenic factor has proangiogenic and proinflammatory effects in the lung via VEGF and VEGF receptor-2

Kazuyo Yamaji-Kegan,1 Qingning Su,1 Daniel J. Angelini,1 Hunter C. Champion,2 and Roger A. Johns1

Departments of 1Anesthesiology and Critical Care Medicine and 2Cardiology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Submitted 4 May 2006 ; accepted in final form 1 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
From a mouse model of hypoxia-induced pulmonary hypertension, we previously found a highly upregulated protein in the lung that we named hypoxia-induced mitogenic factor (HIMF), also known as found in inflammatory zone 1 (FIZZ1), and resistin-like molecule {alpha} (RELM{alpha}). However, the mechanisms of HIMF in the pulmonary vascular remodeling remain unknown. We now demonstrate that HIMF promoted cell proliferation, migration, and the production of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) in pulmonary endothelial cells as well as the production of reactive oxygen species in murine monocyte/macrophage cells. HIMF-induced CD31-positive cell infiltrate in in vivo Matrigel plugs was significantly suppressed by VEGF receptor-2 (VEGFR2) blockade. In ex vivo studies, HIMF stimulated the production of VEGF, MCP-1, and stromal cell-derived factor-1 (SDF-1) in the lung resident cells, and VEGFR2 neutralization significantly suppressed HIMF-induced MCP-1 and SDF-1 production. Furthermore, intravenous injection of HIMF showed marked increase of CD68-positive inflammatory cells in the lungs, and these events were attenuated by VEGFR2 neutralization. Intravenous injection of HIMF also downregulated the expression of VEGFR2 in the lung. These results suggest that HIMF plays critical roles in pulmonary inflammation as well as angiogenesis.

vascular endothelial growth factor; monocyte chemotactic protein-1; vascular endothelial growth factor receptor-2; pulmonary inflammation; hypoxia-induced mitogenic factor; found in inflammatory zone 1; resistin-like molecule {alpha}

PULMONARY HYPERTENSION (PH) is a serious disease of poorly understood etiology characterized by raised pulmonary artery pressure, leading to progressive right-sided heart failure and ultimately death (32). PH results from intimal thickening of small pulmonary resistance arteries that results, at least in part, from endothelial and smooth muscle cell dysfunction and proliferation (41). Increased vascular endothelial cell (EC) proliferation and muscularization of the vasculature are the pathological characteristics of pulmonary vascular remodeling, and it has been demonstrated that this process is associated with hypoxia-induced production of angiogenic factors, inflammatory mediators, and vasoconstrictors.

From a mouse model of hypoxia-induced PH, we previously found a highly upregulated protein that we named hypoxia-induced mitogenic factor (HIMF) (37). A microarray study showed that HIMF gene was significantly upregulated in the lungs of mice that were exposed to hypoxia for 1–4 days. We demonstrated that the recombinant protein of this gene has mitogenic actions and stimulated pulmonary microvascular smooth muscle cell proliferation via an Akt-dependent pathway. HIMF has an identical amino acid sequence to a protein called "found in inflammatory zone 1" (FIZZ1), initially found in a mouse lung allergic inflammation model (10), and resistin-like molecule {alpha} (RELM{alpha}) in adipose tissue (35). HIMF (FIZZ1) is also highly induced in bleomycin-induced lung fibrosis as assessed by cDNA microarray analysis and is found to localize primarily to alveolar epithelial cells by in situ hybridization (16). Furthermore, HIMF (FIZZ1) can induce myofibroblast differentiation in lung fibroblast cultures (18). Our previous study has shown that HIMF is also highly expressed in the perinatal period in cultured embryonic lungs and possesses antiapoptotic properties (43). These studies suggest the potential involvement of HIMF in pulmonary vascular remodeling under pathophysiological conditions. However, the mechanisms and signaling pathways in HIMF-induced biological effects remain unknown.

A particular growth factor that plays an essential role in the cellular adaptation to hypoxia is vascular endothelial growth factor (VEGF), one of the most important prototype angiogenic factors in health and disease. VEGF is considered one of the most specific and potent angiogenic molecules. It has been shown to be essential for vascular development and normal processes such as wound healing and regeneration of the endometrium (6). VEGF expression can become dysregulated in certain pathological conditions such as tumor growth and atherosclerosis (5). It seems conceivable that VEGF is highly involved in the process of hypoxic PH, since in vitro hypoxia rapidly and strongly stimulates the expression of VEGF (22), and recent studies have also shown an increase in VEGF expression in various pulmonary hypertensive disorders, as in advanced pulmonary vascular disease secondary to congenital heart disease (5, 6).

Monocyte chemotactic protein-1 (MCP-1) is a member of the CC chemokine family that plays a critical role in the initiation and progression of inflammation and is also an important mediator of vessel wall remodeling, including angiogenesis. MCP-1 binds to its specific CC chemokine receptor 2 (CCR2), inducing numerous monocyte-mediated proinflammatory signals and monocyte chemotaxis (8). An exclusive perivascular inflammatory cell infiltrate, including monocyte/macrophages, was found in pulmonary arterial hypertension (PAH) patients (40) as well as in a rat model of monocrotaline-induced PH (36). Evidence shows that transient elevation of plasma levels of MCP-1 is associated in the early phase of PH; therefore, this may play a role in the inflammatory response of PAH. Ikeda et al. (13) have suggested that anti-MCP-1 gene therapy attenuated monocrotaline-induced PH in rat. It has also been suggested that MCP-1 is produced by all cellular components of the vessel wall in response to VEGF and hypoxia stimuli (7, 20), and a recent study shows that MCP-1-induced angiogenesis is mediated by VEGF (11).

Angiogenesis and inflammation have been shown to be codependent in many experimental models. Chronic inflammation involves proliferation, migration, and recruitment of tissue and inflammatory cells, which can be extremely damaging to normal tissue. The proliferating tissue contains an abundance of inflammatory cells, angiogenic blood vessels, and derived inflammatory mediators. Under hypoxic conditions, tissue proliferation has outstripped blood vessel growth, which induced further capillary development, and macrophages, for example, are induced to release large quantities of angiogenic factors (15).

In this context, we designed the present study to determine the functional importance of HIMF for new vessel growth and inflammatory response in the lung and to test the following hypotheses: 1) that HIMF stimulates proliferation and migration in pulmonary ECs; 2) that HIMF upregulates proangiogenic and proinflammatory factors; 3) that HIMF recruits inflammatory cells into the lung; and 4) that HIMF promotes inflammation in the lung. We now show that HIMF promotes the production of VEGF and MCP-1 and the infiltration of inflammatory cells into the lung. The ex vivo lung organ culture study suggests that CXCL12/stromal cell-derived factor-1 (SDF-1) is also involved in the intrapulmonary recruitment of circulating cells by HIMF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. RAW 264.7 cells were obtained from American Type Culture Collection (Rockville, MD), and all cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Cells were grown to confluence in DMEM supplemented with 10% FBS. Rat pulmonary microvascular endothelial cells (RPMVECs) were cultured as previously described (3). Culture medium, which consisted of RPMI 1640 with 10% FBS, was changed every other day. Cell integrity was assessed by morphological examination of the cells under phase-contrast microscopy and trypan blue exclusion. All cells were maintained in a humidified incubator at 37°C in a 5% CO2 atmosphere.

Cell proliferation assay. 1 x 104 cells in a 96-well plate were serum-starved overnight (1% BSA, RPMI 1640). Cells were pretreated with VEGF receptor-2 (VEGFR2) inhibitor (100 nM), phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (500 nM), ERK inhibitor U0126 (1 µM), and anti-HIMF antibody (Ab) (1:100) for 30 min and then stimulated with FLAG peptide (Sigma-Aldrich, St. Louis, MO) as a negative control, because the recombinant HIMF is FLAG-tagged at its NH2 terminus, or HIMF protein for 24 h. All inhibitors were purchased from EMD Biosciences (Darmstadt, Germany), and anti-HIMF Ab and recombinant HIMF protein were prepared as described previously (37). Endotoxin level of HIMF protein was <0.1 ng/µg. Viable cells were quantified using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide cell proliferation kit (Roche Applied Science) according to the manufacturer's guidance.

Migration assay. Migration activity was measured using a QCM chemotaxis 96-well cell migration assay kit (Chemicon International, Temecula, CA). Serum-starved cells were trypsinized and resuspended in RPMI 1640 with 1% BSA with or without inhibitors. Cell suspension (100 µl, 3.0 x 104 cells/well) was added to the cell migration chamber. Then, 150 µl of RPMI 1640 with 1% BSA supplemented with HIMF (1–50 nM), FLAG (50 nM), or VEGF (2 ng/ml) was added to the lower chamber and incubated for 20 h. Migrated cells on the lower surface of the membrane were detached and incubated with fluorescent dye (CyQuant GR Dye), and the fluorescence intensity was measured by a microplate reader, FLUOstar OPTIMA (BMG Labtech), using a 485/520-nm filter set.

Determination of cytokine levels in cultured cells. Levels of cytokines were assessed with an ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations. Briefly, 1 x 106 cells were serum-starved overnight and then stimulated with or without HIMF at 37°C in humidified air containing 5% CO2 for 24 h, and each culture media was collected for cytokine determination. For inhibition study, cells were pretreated with wortmannin (500 nM), U0126 (1 µM), or HIMF Ab (1:100) for 30 min before HIMF (50 nM) stimulation. Optical density of the tested samples was compared with the values obtained from serial dilution of respective recombinant cytokine.

Measurement of reactive oxygen species production. Murine monocyte/macrophage cell line RAW 264.7 cells (2.5 x 105 cells in a 24-well plate) were incubated with carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCFDA; Invitrogen), a reliable fluorogenic marker for reactive oxygen species (ROS), and either FLAG or HIMF in a humidified incubator for 1 h. Phorbol 12-myristate 13-acetate (1 µM, Sigma-Aldrich) was used as a positive control for ROS induction. DCFDA reacts with ROS to form the fluorescent product DCF. The fluorescence intensity was measured by a microplate reader using a 485/52-nm filter set. The conversion of DCFDA to DCF was determined by reference to a DCF standard (Sigma-Aldrich), and it was calculated as nmol/2.5 x 105 cells (well)/1 h.

Lung organ culture. Lung organ culture was performed as described previously with minor modifications (17, 33). C57BL/6 mice were euthanized by inhalation of halothane and were bled through the abdominal aorta. The lungs were isolated and infused with 1.5% low-melting temperature agarose solution in organ culture medium (37°C). After the trachea was clamped, the trachea, lungs, and heart were cooled at 4°C for 10 min to solidify the agarose. Once the lung had cooled, complete transverse serial sections (3–5 mm in thickness, 6–8 slices/lung) were gently sliced and mounted on transwell chambers (8-µm pore size; Costar, Cambridge, MA). The slices were cultured in Waymouth's MB752/1 containing 1% heat-inactivated FBS and supplemented with insulin, transferrin, hydrocortisone, sodium selenite, 100 U/ml penicillin G, and 10 µg/ml streptomycin. The slices were incubated at 37°C in a humidified atmosphere of 5% CO2. The lung slices were pretreated with or without VEGFR2 neutralizing Ab (R&D Systems) and then treated with either FLAG or HIMF, and the culture media was harvested at each time point.

In vivo Matrigel plug angiogenesis study and lung morphology study. The formation of new vessels in vivo was evaluated by Matrigel plug assay as described previously with slight modifications (37). C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). Housing and procedures involving experimental animals were approved by the Animal Care and Use Committee of the Johns Hopkins University. Matrigel (400 µl) from BD Biosciences (San Jose, CA) containing FLAG peptide (50 nM) or HIMF (50 nM) was cooled on ice and then injected subcutaneously into the thigh of mice. Mice were killed 7 days after the injection, and the Matrigel plugs with adjacent subcutaneous tissues were harvested for hematoxylin and eosin (HE) staining and immunostaining. To assess the effect of HIMF in the lungs of animals, we injected HIMF protein by two different methods. First, mice were anesthetized with 1–2% isoflurane and etomidate (5–10 mg/kg ip), and a total of 50 µl of Matrigel containing either FLAG peptide (50 nM) or HIMF (50 nM) was injected into the left lungs of the mice transthoracically using transthoracic illumination and a 30-gauge needle. Second, either FLAG protein (200 ng/animal in 100 µl of saline) or HIMF protein (200 ng/animal in 100 µl of saline) was injected into the C57BL/6 mice via the tail veins. For the neutralizing study, mice received VEGFR2 neutralizing Ab (100 µg/animal ip) before either FLAG or HIMF injection. Mice were killed 7 days after the injection, and the lungs were harvested for HE staining and immunostaining. The Matrigel plugs with adjacent subcutaneous tissues or lungs were fixed in 10% or 4% paraformaldehyde, respectively, dehydrated with 30% sucrose, and embedded in optimal cutting temperature compound (Tissue Tek, Sakura Finetechnical). Immunohistostaining for CD31 (PECAM-1, BD Biosciences) and CD68 (Serotec, Raleigh, NC) was performed according to the manufacturer's instructions followed by incubation of secondary antibody [horseradish peroxidase (HRP); HRP-conjugated anti-rat or rabbit IgG at a 1:200 dilution; ABC kit; Vector Laboratory, Burlingame, CA]. Immunoreactivity was visualized with 0.05% diaminobenzidine (Sigma Chemical) as the chromogen. Immunohistochemical negative control sections received identical treatments except for exposure to the primary antibody and showed no specific staining. In Matrigel plug angiogenesis study, CD31-positive cells were counted in four randomly chosen low-power microscopic fields.

Immunoblot for VEGFR2 in the lung. Either FLAG protein (200 ng/animal in 100 µl of saline) or HIMF protein (200 ng/animal in 100 µl of saline) was injected to the C57BL/6 mice via the tail veins, and lung tissues were collected at 1, 4, and 7 days after the injection. Tissue collection, homogenization, and protein electrophoresis were performed as previously described (45). Protein (20 µg) from each sample was subjected to 4–20% precast polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). VEGFR2 expression in the lung was detected with 1:500 dilutions of antibody (Santa Cruz) followed by 1:3,000 dilution of goat-anti-rabbit HRP-labeled antibody (Bio-Rad). ECL substrate kit (Amersham, Piscataway, NJ) was used for the chemiluminescent detection of the signals with autoradiography film (Amersham).

Statistical analysis. All data are given as means ± SD. Differences between groups were assessed by either Student's t-test or ANOVA, followed by Newman post hoc test for multiple comparisons, with P < 0.05 considered to be significant. Unless specifically noted, analysis was performed with n = 4–6/group in a given in vivo assay.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HIMF promotes cell proliferation and migration in pulmonary microvascular ECs. To determine the effect of HIMF on pulmonary microvascular cells, we treated RPMVECs with HIMF or FLAG for 24 h and measured cell proliferation and migration as described in MATERIALS AND METHODS. HIMF stimulated EC proliferation and migration in a dose-dependent manner (Fig. 1, A and C), and these events were significantly suppressed by the preincubation of HIMF Ab, suggesting that HIMF Ab functionally neutralizes these events (Fig. 1, B and D). In our previous study, we showed that HIMF stimulated rat smooth muscle cell proliferation in an Akt-dependent manner (37). In the present study, we also pretreated RPMVECs with angiogenesis-related kinase inhibitors, such as the VEGFR2 inhibitor, the PI3-kinase inhibitor wortmannin, and the ERK inhibitor U0126. The VEGFR2 inhibitor significantly suppressed both HIMF-induced EC proliferation and migration (Fig. 1, B and D). Wortmannin and U0126 also showed significant inhibition of HIMF-induced EC proliferation (Fig. 1B) but did not inhibit HIMF-induced EC cell migration significantly (Fig. 1D), suggesting that these pathways also, at least in part, are involved in HIMF-induced pulmonary EC activation.


Figure 1
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  		Fig. 1. Hypoxia-induced mitogenic factor (HIMF) promotes cell proliferation and migration in rat pulmonary microvascular endothelial cells (RPMVECs). A: dose response of HIMF-induced cell proliferation. RPMVECs were treated with HIMF for 24 h, and cell proliferation levels were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Results were expressed as a percentage of the proliferation index of control (untreated cells) (n = 8 ± SD; *P < 0.05 vs. control). B: inhibition of HIMF-induced cell proliferation by signal inhibitors. RPMVECs were pretreated for 30 min with each reagent and then treated with HIMF (5 nM) for 24 h. Results were expressed as a percentage of the proliferation index of HIMF-treated cells (n = 8; *P < 0.05 vs. control, #P < 0.05 vs. HIMF only). C: dose response of HIMF-induced cell migration. RPMVECs were treated with HIMF for 20 h, and cell migration levels were measured by a modified Boyden chamber assay. Recombinant VEGF (2 ng/ml) was used as a positive control for cell migration. Results were expressed as a percentage of the migration index of control (untreated cells) (n = 8 ± SD; *P < 0.05 vs. control). D: inhibition of HIMF-induced cell migration by signal inhibitors. RPMVECs were pretreated for 30 min with each reagent and then treated with HIMF (10 nM) for 20 h. Results were expressed as a percentage of the migration index of HIMF-treated cells (n = 8 ± SD; #P < 0.05 vs. HIMF only). VEFGR2, vascular endothelial growth factor receptor-2; Wort., wortmannin; Ab, antibody.

 
HIMF promotes the production of proangiogenic and proinflammatory mediators in vitro. VEGF is known as a strong mediator that promotes EC proliferation, migration, angiogenesis, and vascular permeability, and recent studies have suggested that endothelial MCP-1 is induced by VEGF and seems to participate in angiogenesis (46). To examine the effect of HIMF on angiogenesis-related mediators in pulmonary ECs, VEGF and MCP-1 levels in the cell culture media were measured after we treated RPMVECs with HIMF or FLAG for 24 h. HIMF stimulated both VEGF and MCP-1 production in a dose-dependent manner (Fig. 2, A and B). In addition, HIMF-induced VEGF and MCP-1 production was significantly suppressed by the preincubation of wortmannin and U0126, suggesting that this is PI3-kinase and ERK pathway dependent. Wortmannin, U0126, and HIMF Ab alone had no effect on VEGF or MCP-1 production (data not shown). It has been suggested that induction of VEGF expression and promotion of angiogenesis are mediated by ROS. It appears that cellular response to ROS is mediated by activation of Ras/raf/p42/p44 MAPK and PI3K/Akt pathways to induce VEGF expression (44). Recruitment of leukocytes into the lung air spaces or the vessel wall itself can have significant impact on the function and structure of lung blood vessels. Activated macrophages and/or neutrophils can release a variety of factors, including ROS, capable of directly causing vasoconstriction (31). In this context, we examined the effect of HIMF on ROS production in murine monocyte/macrophage cell line RAW 264.7 cells. When cells were treated with HIMF, we observed a significant increase of ROS production, and it reached a maximum level at 10 nM HIMF treatment (Fig. 2C).


Figure 2
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  		Fig. 2. HIMF promotes production of proangiogenic, proinflammatory mediators and reactive oxygen species (ROS) in vitro. A: dose response of HIMF-induced VEGF production and inhibition of HIMF-induced VEGF production by signal inhibitors. RPMVECs were pretreated for 30 min with no drugs, wortmannin (500 nM), U0126 (1 µM), or HIMF Ab (1:100), and then treated with HIMF (50 nM) for 24 h. VEGF release was measured by ELISA (n = 4 ± SD; *P < 0.05 vs. control, #P < 0.05 vs. 50 nM HIMF). B: dose response of HIMF-induced monocyte chemotactic protein-1 (MCP-1) production and inhibition of HIMF-induced MCP-1 production by signal inhibitors. RPMVECs were pretreated for 30 min with or without drugs and then treated with HIMF (50 nM) for 24 h. MCP-1 release was measured by ELISA (n = 4 ± SD; *P < 0.05 vs. control, #P < 0.05 vs. HIMF 50 nM). C: HIMF stimulates ROS production. RAW 264.7 cells were treated with carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCFDA) and then treated with FLAG (10 nM) or HIMF (1–10 nM) for 1 h. Phorbol 12-myristate 13-acetate (PMA, 1 µM) was used as a positive control for ROS production. ROS levels were expressed as DCF production (nmol per well/1 h) (n = 8 ± SD; **P < 0.01 vs. control).

 
HIMF upregulates VEGF, MCP-1, and SDF-1 expression in the organotypic lung slices. To examine the effects of HIMF on the lung resident cells, especially alveolar epithelial cells, we used an ex vivo lung organ culture model, which is a blood-free and well-established method. The additional advantage in using this organ culture system is that the effect of HIMF on the lung slices could be estimated in a model similar to the in vivo model. Because the alveolar structure of the lung slice is kept intact, the cells do not lose their local contacts, and the differentiated state of each cell is maintained by cell-matrix contact, cell-cell contact, and local soluble factors.

The lung slices were cultured with media that contains either HIMF (10 nM) or FLAG (10 nM) for 24 h, and the lung organ culture media was collected for the determination of angiogenesis-related mediators. HIMF treatment (10 nM, 24 h) significantly increased VEGF and MCP-1 production in the lung organ culture media (Fig. 3, A and B, respectively). HIMF-stimulated MCP-1 production was attenuated to the basal level with pretreatment of the VEGFR2 neutralizing Ab, and these results are in accordance with Figs. 1 and 2.


Figure 3
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  		Fig. 3. HIMF upregulates proangiogenic factors in the organotypic lung slices. HIMF-induced VEGF (A), MCP-1 (B), and stromal cell-derived factor (SDF)-1 (C) productions in the lung organ culture slices. Lung slices were treated with FLAG (10 nM), HIMF (10 nM), or HIMF (10 nM) and VEGFR2 neutralizing Ab (150 ng/ml) for 24 h. The amount of VEGF, MCP-1, and SDF-1 released into the media was measured by ELISA (n = 4 ± SD, *P < 0.05 or **P < 0.01 vs. control, #P < 0.05 vs. HIMF only). This experiment was repeated 3 times with similar results. Cont, control.

 
Other chemokines such as CXCL12/SDF-1 are involved in monocyte recruitment to the hypoxic vessel wall. Monocytes and macrophages respond to the chemotactic effects of SDF-1 via the expression of the CXCR4 receptor. Hypoxia has been shown to significantly upregulate expression of SDF-1 in resident fibroblasts and endothelial cells (34). For this reason, we examined the effect of HIMF on SDF-1 production in the lung resident cells. Treatment with HIMF resulted in a significant increase of SDF-1 in the lung resident cells, and this effect was completely inhibited by the addition of VEGFR2 neutralizing Ab (Fig. 3C). However, in our present study, we did not observe SDF-1 production under any conditions in RPMVECs (data not shown). The VEGFR2 Ab itself had no effect on VEGF, MCP-1, and SDF-1 production (data not shown). These data suggest the possibility that circulating cells contribute to promoting HIMF-stimulated lung inflammation and vascular remodeling.

VEGFR2 regulates HIMF-induced vascular cell infiltration. VEGF isoforms exert their biological effects through interaction with two tyrosine kinase receptors, VEGFR2 (also known as KDR human homolog or Flk-1 murine homolog) and VEGFR1 (also known as Flt-1). Several studies have suggested VEGFR2 is the crucial receptor for transmitting cellular signals for the proliferation, differentiation, and migration of ECs, and VEGFR2-deficient mice show dramatic defects in angiogenesis and hematopoiesis (27). Since HIMF significantly increased the vascular tube formation in Matrigel plug in vivo in our previous study (37), we examined the effect of VEGFR2 inhibitor on HIMF-induced vascular cell infiltration in vivo. In this study, either FLAG or HIMF containing Matrigel plugs were injected subcutaneously in the animals. Seven days after injection, Matrigel plugs were harvested and stained with HE (Fig. 4, A–C), revealing that HIMF promoted a marked increase of cell infiltration in the Matrigel plugs (Fig. 4B) compared with the control (Fig. 4A). This event was suppressed by the addition of VEGFR2 inhibitor (Fig. 4C). We also assessed EC infiltration of the plugs by immunohistochemical analysis of CD31-positive cells (Fig. 4, D–F). Quantitative analyses of histological sections revealed that plugs containing HIMF displayed a significantly higher density of CD31-positive cells compared with controls, and this stimulatory effect was significantly decreased by the addition of VEGFR2 inhibitor (Fig. 4G). The VEGFR2 inhibitor alone did not affect vascular cell infiltration (data not shown). These results suggest that VEGFR2 and its related molecules regulate HIMF-stimulated vascular formation in vivo.


Figure 4
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  		Fig. 4. VEGFR2 inhibitor blocks HIMF-induced cell infiltrate in the Matrigel plugs in vivo. An in vivo Matrigel plug assay was performed to evaluate the HIMF-induced blood vessel formation. Hematoxylin and eosin (HE) staining (A–C, scale bars: 100 µm) and immunostaining for CD31 (D–F, scale bars: 50 µm) are shown. A and D: Matrigel plugs with FLAG (50 nM). B and E: Matrigel plugs with HIMF (50 nM). C and F: Matrigel plugs with HIMF (50 nM) and VEGFR2 inhibitor (100 nM). G: the frequency of CD-31 positive cells in four low power fields was determined for each Matrigel plug. Data were presented as fold increase of CD31-positive cells relative to the control (n = 4 ± SD; *P < 0.05 vs. control, #P < 0.05 vs. HIMF only). This experiment was repeated 3 times with similar results. H and I: HIMF promotes cell recruitment in the lung. Either FLAG (50 nM/50 µl Matrigel plugs, H) or HIMF (50 nM/50 µl Matrigel plugs, I) were injected in the lungs of mice directly, and the lungs were harvested for lung histology analysis (HE staining, scale bars: 20 µm).

 
HIMF promotes cell recruitment in the lung. Since we previously confirmed that HIMF protein expression was upregulated only in the lungs of the mice under hypoxic conditions (37), we examined the effect of HIMF in pulmonary vasculature in vivo. In the present study, we injected either FLAG or HIMF with Matrigel into the lung directly so that we could examine the location of the HIMF protein in the area where it was injected into the lung. In this model, Matrigel appeared pink-to-light reddish in color with HE staining (Fig. 4H), and a dramatic number of cells was recruited in the area of Matrigel containing HIMF (Fig. 4I), especially in the airway epithelial cell area compared with control lungs (Fig. 4H).

Neutralization of VEGFR2 attenuates HIMF-induced inflammation in the lung. To examine the effect of HIMF in the mouse lung, we instilled HIMF via tail vein. When HIMF was administered intravenously, marked increase of capillary leakage was observed compared with the control lung (Fig. 5A). Lung histology analysis 7 days after the administration of HIMF or FLAG protein revealed a dramatic increase of circulating cells in the lung in response to HIMF (Fig. 5C). HIMF-induced widespread intra-alveolar edema suggests that increased pulmonary vascular permeability in the early stage of acute lung injury may be induced.


Figure 5
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  		Fig. 5. Intravenous injection of HIMF promotes inflammation in the lung. A: inflated left lungs showing marked increase of blood vessels and inflammation with intravenous injection of HIMF compared with the control. Representative lung histology demonstrates marked inflammation in responses to HIMF characterized by abundant interstitial circulating cells and edema relative to control lungs. VEGFR2 neutralization attenuated HIMF-induced lung inflammation. B–D: HE staining (scale bars: 100 µm); E–G: CD68-positive cells (arrows, scale bars: 50 µm).

 
In the present study, we demonstrated that HIMF-induced MCP-1 and SDF-1 production in the lung resident cells (Fig. 3, B and C, respectively) was significantly suppressed by VEGFR2 blockade as well as HIMF-induced EC proliferation and migration in RPMVECs (Fig. 1, B and D, respectively). We also demonstrated that VEGFR2 blockade suppressed HIMF-induced cell infiltrate in the in vivo Matrigel plugs (Fig. 4, C and F, respectively). Thus we examined whether neutralization of VEGFR2 would suppress HIMF-induced pulmonary inflammation and injury in the lung. VEGFR2 neutralization significantly attenuated HIMF-induced inflammation with a decreased prevalence of edema in tissue (Fig. 5D), achieving a level of inflammation comparable to that observed in control lungs (Fig. 5B). The VEGFR2 Ab alone had no appreciable effect on lung histology (data not shown).

It is well known that tissue macrophages are potent producers of proangiogenic factors and thus are important contributors to new blood vessel formation (24, 28). Chronic inflammation involves proliferation, migration, and recruitment of tissue and inflammatory cells, which can be extremely damaging to normal tissue. It has also been suggested that these areas contain an abundance of inflammatory cells, angiogenic blood vessels, and derived inflammatory mediators. Macrophages, especially, are induced to release large quantities of angiogenic factors under hypoxic conditions (24). In this context, we examined whether HIMF would stimulate monocyte/macrophage recruitment in the lung, and immunohistochemical analysis for CD68-positive cells was performed. In the HIMF-injected lungs, a large number of CD68-positive cells was detected (Fig. 5F) compared with the control lungs (Fig. 5E), and this effect was diminished by neutralization of VEGFR2 (Fig. 5G).

Effect of HIMF on VEGFR2 expression in the lung. There is evidence that VEGFR2 expression in the lung is decreased by various stimuli that promote lung inflammation, such as prolonged hypoxia (25), LPS (14), and oxidant stress (42). Thus we examined the effect of HIMF on VEGFR2 expression in the lung at different time points after intravenous injection of HIMF or FLAG. As seen in Fig. 6, expression of VEGFR2 was significantly decreased in the lung at 1, 4, and 7 days after HIMF injection when compared with the control lungs.


Figure 6
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  		Fig. 6. Effect of HIMF on VEGFR2 expression in the lung. Shown is VEGFR2 expression in the lungs of mice that were injected with HIMF or FLAG for 1, 4, and 7 days. The bands were identified at 230 kDa (n = 4 ± SD; *P < 0.05 or **P < 0.01 vs. control).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of this study is that HIMF upregulates proinflammatory mediators in the lung in relation to its effects on vascular remodeling. Our results suggest that HIMF activates lung resident cells by stimulating VEGF and MCP-1 production, which are important factors for the early stage of pulmonary vascular remodeling. Our data also suggest that HIMF promotes inflammation by recruiting inflammatory cells such as macrophages in the lung.

Macrophages represent an important component of the leukocytic infiltrate in a majority of malignant tumors. They are thought to originate from peripheral blood monocytes that are recruited into the tumor from the circulation rather than from resident macrophages present in tissue before the tumor developed, specifically via hypoxia-regulated signaling pathways. It is well known that tissue macrophages are potent producers of proangiogenic and proinflammatory factors (24). Several recent studies document the enhanced expression of proinflammatory mediators and increased numbers of macrophages and neutrophils in the lungs of mice and rats exposed to hypoxia. Minamino et al. (21) demonstrated that mice exposed to acute hypoxia exhibited a marked induction of proinflammatory cytokines and chemokines in association with increased numbers of macrophages and neutrophils. It has been suggested that the hypoxia-induced accumulation of macrophages appeared to be specific for the pulmonary circulation because no macrophage recruitment was noted in the systemic circulation (34). There is evidence from studies with alternatively activated macrophages that suggest induction of HIMF (FIZZ1) expression may be under the influence of type 2 cytokines (26, 29). Liu et al. (19) have suggested that Th2 cytokines IL-4 and IL-13 are potent inducers of alveolar type II epithelial cell HIMF (FIZZ1) expression via STAT6 pathway. A variety of factors are involved in the recruitment of monocytes from the blood stream to the specific tissue sites. Chemokines are known to induce recruitment of monocytes from the blood stream into tissues. MCP-1 is one of the most potent chemokines that attract and activate monocytes, and this molecule has been most implicated in monocyte recruitment in tumors and other inflammatory disease processes (24). Ikeda et al. (13) demonstrated that a dominant negative inhibitor of MCP-1 significantly reduced the progression of monocrotaline-induced PH, including the inhibition of mononuclear cell infiltration into the lung.

It has been suggested that, within the pulmonary circulation, ROS as well as hypoxia upregulate the expression of the molecules that induce inflammatory cell recruitment. Exposure to hypoxia elevates pulmonary arterial pressure, which results in activating proinflammatory responses such as P-selectin upregulation, in ECs of the lung. This response seems to occur by ROS generated by mitochondria (12). ROS are also known to be involved in VEGF signaling linked to angiogenic responses. It appears that cellular response to ROS is mediated mainly by activation of the Ras/raf/p42/p44 MAPK, Rac/MAPK kinase 1/JNKs, and Rac/PAC/p38/MAPK signaling, resulting in activation of many downstream targets, such as Sp1, AP-1, NF-{kappa}B, and Stat3, which are important for regulation of VEGF expression. ROS also activate the PI3-kinase/Akt pathway, which leads to hypoxia-inducible factor (HIF)-1 induction and VEGF upregulation. Additionally, ROS function as downstream mediators of angiogenic signaling by VEGF/VEGFR2 (44). A recent study that suggests that HIMF upregulates VEGF in mouse epithelial cells via the PI3-kinase/Akt-NF-{kappa}B-dependent pathway (39) is consistent with our observations and is in accordance with our present study that ROS are involved in HIMF downstream signaling. We also demonstrated that the HIMF-induced VEGF production in RPMVECs was significantly attenuated by the preincubation of PI3-kinase inhibitor and the ERK inhibitor, indicating that these signaling pathways are involved in HIMF downstream signaling in pulmonary ECs.

The lung is unique in its double sources of perfusion from the pulmonary and systemic circulation, and the pulmonary vasculature seems to have much more limited capacity for angiogenesis than the other systemic arteries (23). Recent data of Davie and colleagues (4) have demonstrated that upregulated VEGF expression in the pulmonary artery adventitia of chronically hypoxic animals is associated with increased neovascularization and monocyte accumulation. In the present study, HIMF stimulated significant proangiogenic and chemotactic factors in the lung resident cells, suggesting that HIMF has the potential to contribute to the initiation of pulmonary vascular remodeling by stimulating these lung resident cells followed by the recruitment of circulating cells. The fact that pulmonary HIMF expression was noted in the bronchial epithelial cells, alveolar type II cells, and vascular endothelial cells, in an allergic model of pulmonary inflammation (10), and in the hypoxic model of pulmonary hypertension (37), also support our findings that HIMF activates those lung resident cells.

In the present study, HIMF also stimulated SDF-1 production in the lung resident cells. Monocytes and macrophages respond to the chemotactic effects of SDF-1 via the expression of the C-X-C chemokine receptor 4 (CXCR4). Oxygen availability is a determinant factor in the settings of chemotactic responsiveness to SDF-1. Hypoxia has been shown to significantly upregulate expression of SDF-1 as well as VEGF via a (HIF-{alpha})-dependent pathway in resident fibroblasts and ECs (2). Hypoxia also induces high expression of CXCR4 via HIF-{alpha} in different cell types (monocytes, macrophages, ECs, and cancer cells), which is paralleled by increased chemotactic responsiveness to its specific ligand. HIMF has been shown to be upregulated by chronic hypoxia in vivo, and the 3' region of the HIMF gene contains a binding site for HIF-1{alpha} and HIF-2{alpha} (37). We also have reported that HIMF is expressed in the developing lung, and HIMF protein colocalizes spatially and temporally with HIF-2{alpha}, indicating that the regulation of HIMF expression is under the control of HIF-2{alpha} (43). Brusselmans et al. (1) have demonstrated that heterozygous deficiency of HIF-2{alpha} protects mice against PH and right ventricular dysfunction during prolonged hypoxia. These findings indicate that HIF-2{alpha} might be one of the key upstream regulators of HIMF expression, and both HIMF and HIF-2{alpha} might play a pivotal role in hypoxia-induced pulmonary vascular remodeling.

In the present study, we also demonstrated that HIMF caused downregulation of VEGFR2 expression in the lung. There is evidence that VEGFR2 expression in the lung is decreased in various lung inflammation models (25, 38, 42). In the recent study, the expression of HIMF in the lung was upregulated in a LPS-induced acute lung injury model (38). Upregulation of HIMF expression in LPS-induced acute lung injury indicates that HIMF plays an important role in lung inflammation, which is in accordance with our present findings. The downregulated expression of VEGFR2 may be due to increase in proinflammatory cytokines in the present study.

RELMbeta represents the most related human homolog of HIMF in rodents. A recent study has suggested that human RELMbeta also has mitogenic action on human lung cells, and this protein may contribute to pulmonary vascular remodeling and fibrotic lung disease (30). Considering the limitations of current treatment of PH (9), inhibition of HIMF might be considered to prevent or reduce lung inflammation as well as hypoxia-induced PH. The finding of this study may ultimately result in novel therapies designed to attenuate the HIMF biological axis and lead to better intervention in the prevention and treatment of hypoxia-induced pulmonary vascular remodeling as well as lung inflammation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-39706 (to R. A. Johns).


   ACKNOWLEDGMENTS
 
We thank John Skinner for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Johns, Ross 361, The Johns Hopkins Medical Institutions, 720 Rutland Ave., Baltimore, MD 21205 (e-mail: rajohns{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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