SP-D loaded PLGA nanoparticles as drug delivery system for prevention and treatment of premature infant's lung diseases.

Preterm infants, particularly those who born between 23 and 28 weeks' gestation, suffer from a very high incidence of respiratory distress syndrome (RDS) related to pulmonary immaturity and inability to make Pulmonary Surfactant (PS). These infants are supported by the use of oxygen, ventilators, and routine administration of surfactant replacement. The currently commercial surfactant replacement therapies do not contain hydrophilic surfactant proteins such as Surfactant Protein D (SP-D). These proteins have a key role in the innate lung host defense, thus the development of a sustained release vehicle that provides SP-D for long periods in preterm infants' lungs would exploit the therapeutic potential of SP-D and other pulmonary medications. The proposed SP-D delivery system is based on nanoparticles (NPs) composed of poly (lactic acid-co-glycolic acid) (PLGA), a biodegradable, FDA approved biopolymer. The resulted NPs were spherical with high Zeta potential value, were not toxic to A-549 lungs cells, and did not induce any inflammatory response in mouse's lungs for short and long-term periods. Moreover, SP-D released from NPs showed biological activity for several days and in vivo release experiment of SP-D loaded NPs revealed that SP-D was released from NPs in mouse lungs with different NPs delivery doses.

Klf5 controls bone marrow homing of stem cells and progenitors through Rab5-mediated ß1/ß2-integrin trafficking.

Krüppel-like factor 5 regulates pluripotent stem cell self-renewal, but its role in somatic stem cells is unknown. Here we show that Krüppel-like factor 5-deficient haematopoietic stem cells and progenitors fail to engraft after transplantation. This haematopoietic stem cell and progenitor defect is associated with impaired bone marrow homing and lodging and decreased retention in bone marrow, and with decreased adhesion to fibronectin and expression of membrane-bound ß1/ß2-integrins. In vivo-inducible gain-of-function of Krüppel-like factor 5 in haematopoietic stem cells increases haematopoietic stem cell and progenitor adhesion. The expression of Rab5 family members, mediators of ß1/ß2-integrin recycling in the early endosome, is decreased in Klf5(Δ/Δ) haematopoietic stem cells and progenitors. Krüppel-like factor 5 binds directly to the promoter of Rab5a/b, and overexpression of Rab5b rescues the expression of activated ß1/ß2-integrins, adhesion and bone marrow homing of Klf5(Δ/Δ) haematopoietic stem cells and progenitors. Altogether, these data indicate that Krüppel-like factor 5 is indispensable for adhesion, homing, lodging and retention of haematopoietic stem cells and progenitors in the bone marrow through Rab5-dependent post-translational regulation of ß1/ß2 integrins.

Skeletal demineralization and fractures caused by fetal magnesium toxicity.

Two surviving female infants, born from a triplet pregnancy at 30 weeks gestation, were noted to have severe osteopenia and multiple fractures diagnosed at 20 days of age. Their mother had been treated for preterm labor with intravenous magnesium sulfate from week 22 until their birth at 30 weeks gestation. At birth, the triplets exhibited craniotabes with enlarged fontanelles and sutures. All developed Respiratory Distress Syndrome (RDS) and the two surviving infants required prolonged respiratory support. Serum calcium and phosphate levels were normal and alkaline phosphatase levels were increased. The infants were treated with supplements of calcium and phosphorous, with resultant healing of the multiple fractures without deformity. Fetal magnesium toxicity impairs bone mineralization and can lead to serious bone demineralization that may cause fractures in the newborn period that complicate recovery from respiratory disease. Early recognition and treatment may minimize complications related to osteopenia caused by fetal magnesium toxicity.

Stage-specific regulation of respiratory epithelial cell differentiation by Foxa1.

Foxa1 is a member of the winged helix family of transcription factors that is expressed in epithelial cells of the conducting airways and in alveolar type II cells of the lung. To determine the role of Foxa1 during lung morphogenesis, histology and gene expression were assessed in lungs from Foxa1-/- gene-targeted mice from embryonic day (E) 16.5 to postnatal day (PN) 13. Deletion of Foxa1 perturbed maturation of the respiratory epithelium at precise times during lung morphogenesis. While dilatation of peripheral lung saccules was delayed in Foxa1-/- mice at E16.5, sacculation was unperturbed later in development (E17.5-E18.5). At PN5, alveolarization was markedly delayed in Foxa1-/- mice; however, by PN13 lung histology was comparable to wild-type controls. Clara cell secretory protein (CCSP), prosurfactant protein (SP)-C, and SP-B protein content and immunostaining were decreased in Foxa1-/- mice between E16.5 and E18.5 but normalized after birth. Timing and sites of expression of thyroid transcription factor-1, Foxj1, and beta-tubulin were unaltered in lungs of Foxa1-/- mice. In vitro, Foxa1 regulated the activity of CCSP and SP-A, SP-B, SP-C, and SP-D promoters as assessed by luciferase reporter assays in HeLa, H441, and MLE15 cells. Although Foxa1 regulates respiratory epithelial differentiation and structural maturation of the lung at precise developmental periods, the delay in maturation is subsequently compensated at times to enable respiratory function and restore normal lung structure after birth.

Interactions between pulmonary surfactant and alveolar macrophages in the pathogenesis of lung disease.

This article reviews recent findings regarding interrelationships between the pulmonary surfactant system and alveolar macrophages in the pathogenesis of human lung diseases, including pulmonary alveolar proteinosis, pulmonary infection, inflammation and obstructive lung diseases. Various components of pulmonary surfactant interact with alveolar macrophages in the modulation of innate host defense responses of the lung. In a reciprocal manner, alveolar macrophages regulate surfactant homeostasis, influencing rates of surfactant protein and phospholipid clearance and catabolism. Increasing evidence suggests that the complex interactions between the surfactant system and the alveolar macrophage are intrinsically linked to development of various lung diseases.

Transient induction of TGF-alpha disrupts lung morphogenesis, causing pulmonary disease in adulthood.

Clinical studies have associated increased transforming growth factor (TGF)-alpha and EGF receptor with lung remodeling in diseases including bronchopulmonary dysplasia (BPD). BPD is characterized by disrupted alveolar and vascular morphogenesis, inflammation, and remodeling. To determine whether transient increases in TGF-alpha are sufficient to disrupt postnatal lung morphogenesis, we utilized neonatal transgenic mice conditionally expressing TGF-alpha. Expression of TGF-alpha from postnatal days 3 to 5 disrupted postnatal alveologenesis, causing permanent enlargement of distal air spaces in neonatal and adult mice. Lung volume-to-body weight ratios and lung compliance were increased in adult TGF-alpha transgenic mice, whereas tissue and airway elastance were reduced. Elastin fibers in the alveolar septae were fragmented and disorganized. Pulmonary vascular morphogenesis was abnormal in TGF-alpha mice, with attenuated and occasionally tortuous arterial branching. The ratios of right ventricle weight to left ventricle plus septal weight were increased in TGF-alpha mice, indicating pulmonary hypertension. Electron microscopy showed gaps in the capillary endothelium and extravasation of erythrocytes into the alveolar space of TGF-alpha mice. Hemorrhage and inflammatory cells were seen in distal air spaces at 1 mo of age. In adult TGF-alpha mice, alveolar remodeling, nodules, proteinaceous deposits, and inflammatory cells were seen. Immunostaining for pro-surfactant protein C showed that type II cells were abundant in the nodules, as well as neutrophils and macrophages. Trichrome staining showed that pulmonary fibrosis was minimal, apart from areas of nodular remodeling in adult TGF-alpha mice. Transient induction of TGF-alpha during early alveologenesis permanently disrupted lung structure and function and caused chronic lung disease.

VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice.

To determine whether increased levels of VEGF disrupt postnatal lung formation or function, conditional transgenic mice in which VEGF 164 expression was enhanced in respiratory epithelial cells were produced. VEGF expression was induced in the lungs of VEGF transgenic pups with doxycycline from postnatal day 1 through 2 and 6 wk of age. VEGF levels were higher in bronchoalveolar lavage fluid (BALF) and lung homogenates of VEGF transgenic mice compared with endogenous VEGF levels in controls. Neonatal mortality was increased by 50% in VEGF transgenic mice. Total protein content in BALF was elevated in VEGF transgenic mice. Surfactant protein B protein expression was unaltered in VEGF transgenic mice. Although postnatal alveolar and vascular development were not disrupted by VEGF expression, VEGF transgenic mice developed pulmonary hemorrhage, alveolar remodeling, and macrophage accumulation as early as 2 wk of age. Electron microscopy demonstrated abnormal alveolar capillary endothelium in the VEGF transgenic mice. In many locations, the endothelium was discontinuous with segments of attenuated endothelial cells. Large numbers of hemosiderin-laden macrophages and varying degrees of emphysema were observed in adult VEGF transgenic mice. Overexpression of VEGF in the neonatal lung increased infant mortality and caused pulmonary hemorrhage, hemosiderosis, alveolar remodeling, and inflammation.

Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes.

To investigate the role of an activated K-Ras gene in the initiation and maintenance of lung adenocarcinomas, we developed transgenic mice that express murine K-Ras4b(G12D) under the control of doxycycline in type II pneumocytes. Focal proliferative lesions of alveolar type II pneumocytes were observed as early as seven days after induction with doxycycline; after two months of induction, the lungs contained adenomas and adenocarcinomas, with focal invasion of the pleura at later stages. Removal of doxycycline caused a rapid fall in levels of mutant K-Ras RNA and concomitant apoptotic regression of both the early proliferative lesions and the tumors. Tumor burden was dramatically decreased by three days after withdrawal, and tumors were undetectable after one month. When similar experiments were performed with animals deficient in either the p53 gene or the Ink4A/Arf locus, tumors arose more quickly (within one month of exposure to doxycycline) and displayed more obvious histological features of malignancy; nevertheless, these tumors also regressed rapidly when the inducer was removed, implying that continued production of mutant K-Ras is necessary to maintain the viability of tumor cells in the absence as well as the presence of tumor suppressor genes. We also show that the appearance and regression of these pulmonary tumors can be readily monitored in anesthetized transgenic animals by magnetic resonance imaging.

Immunolocalization of sonic hedgehog (Shh) in developing mouse lung.

Expression of sonic hedgehog (Shh) is required for normal development of the lung during embryogenesis. Loss of Shh expression in mice results in tracheoesophageal fistula, lung hypoplasia, and abnormal lung lobulation. To determine whether Shh may play a role later in lung morphogenesis, immunostaining for Shh was performed in mouse lung from embryonic day (E) 10.5 to postnatal day (PD) 24. Shh was detected in the distal epithelium of the developing mouse lung from E10.5 to E16.5. From E16.5 until PD15, Shh was present in epithelial cells in both the peripheral and conducting airways. Although all cells of the developing epithelium uniformly expressed Shh at E10.5, Shh expression was restricted to subsets of epithelial cells by E16.5. Between E16.5 and PD15, non-uniform Shh staining of epithelial cells was observed in the conducting airways in a pattern consistent with the distribution of non-ciliated bronchiolar cells (i.e., Clara cells) and the Clara cell marker CCSP. Shh did not co-localize with hepatocyte nuclear factor/forkhead homologue-4 (HFH-4), beta-tubulin, or with the presence of cilia. These results support the concept that Shh plays a distinct regulatory role in the lung later in morphogenesis, when it may influence formation or cytodifferentiation of the conducting airways.

Aquaporin 5-deficient mouse lungs are hyperresponsive to cholinergic stimulation.

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5(-/-)) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5(+/+) mice, Aqp5(-/-) mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5(-/-) mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5(-/-) mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.

Surfactant protein D enhances clearance of influenza A virus from the lung in vivo.

Mice lacking surfactant protein surfactant protein D (SP-D(-/-)) and wild-type mice (SP-D(+/+)) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D(-/-) mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV.

GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1.

GM-CSF gene targeted (GM(-/-)) mice are susceptible to respiratory infections and develop alveolar proteinosis due to defects in innate immune function and surfactant catabolism in alveolar macrophages (AMs), respectively. Reduced cell adhesion, phagocytosis, pathogen killing, mannose- and Toll-like receptor expression, and LPS- or peptidoglycan-stimulated TNFalpha release were observed in AMs from GM(-/-) mice. The transcription factor PU.1 was markedly reduced in AMs of GM(-/-) mice in vivo and was restored by selective expression of GM-CSF in the lungs of SPC-GM/GM(-/-) transgenic mice. Retrovirus-mediated expression of PU.1 in AMs from GM(-/-) mice rescued host defense functions and surfactant catabolism by AMs. We conclude that PU.1 mediates GM-CSF-dependent effects on terminal differentiation of AMs regulating innate immune functions and surfactant catabolism by AMs.

Fetal monkey surfactants after intra-amniotic or maternal administration of betamethasone and thyroid hormone.

OBJECTIVE: To compare direct intra-amniotic injection of betamethasone and thyroxine (T4) with maternal treatment and controls for accelerating pulmonary surfactant production. METHODS: Twelve pregnant monkeys (Macaca mulatta) on gestational day 125 (term 165 +/- 10 days) had surfactant protein A and B concentrations measured in amniotic fluid. In four controls, normal saline was injected into the amniotic fluid; four others (intra-amniotic) received intra-amniotic betamethasone (1 mg) and T4 (60 microg); and in four others (maternal), the dam was given betamethasone (12 mg) intramuscularly, repeated in 24 hours, plus TRH (400 microg) intravenously, repeated every 6 hours for 24 hours. Seventy-two hours after the initial amniocentesis, a hysterotomy was performed and fetal tissue and amniotic fluid harvested for determination of surfactant protein A and B concentrations and immunohistochemical staining for surfactant protein A. RESULTS: Amniotic fluid surfactant protein A was higher with intra-amniotic injection than with maternal treatment (P <.04) or controls (P =.07). Amniotic fluid surfactant protein B was higher in the intra-amniotic group than in controls (P =.06). Immunohistochemical staining for surfactant protein A in the lung tissue was increased in the intra-amniotic group compared with controls (0.145 +/- 0.01 versus 0.097 +/- 0.001, percent positive staining for surfactant protein A cells per lung tissue cells; P <.03). Birth weight was greater in the intra-amniotic group compared with the maternal group (P <.03) although not different from the controls. Finally, gut motility and the presence of formed meconium were increased in the intra-amniotic group compared with the other groups (P <.05). CONCLUSION: Intra-amniotic injection of betamethasone and T4 enhanced lung (and possibly intestinal) maturation of the preterm rhesus fetal monkey compared with maternal injections.

SP-D and GM-CSF regulate surfactant homeostasis via distinct mechanisms.

Both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF) influence pulmonary surfactant homeostasis, with the deficiency of either protein causing marked accumulation of surfactant phospholipids in lung tissues and in the alveoli. To assess whether the effects of each gene were mediated by distinct or shared mechanisms, surfactant homeostasis and lung morphology were assessed in 1) double-transgenic mice in which both SP-D and GM-CSF genes were ablated [SP-D(-/-),GM(-/-)] and 2) transgenic mice deficient in both SP-D and GM-CSF in which the expression of GM-CSF was increased in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were markedly increased in SP-D(-/-),GM(-/-) mice, with the effects of each gene deletion on surfactant Sat PC pool sizes being approximately additive. Expression of GM-CSF in lungs of SP-D(-/-),GM(-/-) mice corrected GM-CSF-dependent abnormalities in surfactant catabolism but did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(-/-) mice, degradation of [(3)H]dipalmitoylphosphatidylcholine by alveolar macrophages from the SP-D(-/-) mice was normal. The emphysema and foamy macrophage infiltrates characteristic of SP-D(-/-) mice were similar in the presence or absence of GM-CSF. Taken together, these findings demonstrate the distinct roles of SP-D and GM-CSF in the regulation of surfactant homeostasis and lung structure.

The collagen-like region of surfactant protein A (SP-A) is required for correction of surfactant structural and functional defects in the SP-A null mouse.

Pulmonary surfactant isolated from gene-targeted surfactant protein A null mice (SP-A(-/-)) is deficient in the surfactant aggregate tubular myelin and has surface tension-lowering activity that is easily inhibited by serum proteins in vitro. To further elucidate the role of SP-A and its collagen-like region in surfactant function, we used the human SP-C promoter to drive expression of rat SP-A (rSPA) or SP-A containing a deletion of the collagen-like domain (DeltaG8-P80) in the Clara cells and alveolar type II cells of SP-A(-/-) mice. The level of the SP-A in the alveolar wash of the SP-A(-/-,rSP-A) and SP-A(-/-,DeltaG8-P80) mice was 6.1-and 1.3-fold higher, respectively, than in the wild type controls. Tissue levels of saturated phosphatidylcholine were slightly reduced in the SP-A(-/-,rSP-A) mice compared with SP-A(-/-) littermates. Tubular myelin was present in the large surfactant aggregates isolated from the SP-A(-/-,rSP-A) lines but not in the SP-A(-/-,DeltaG8-P80) mice or SP-A(-/-) controls. The equilibrium and minimum surface tensions of surfactant from the SP-A(-/-,rSP-A) mice were similar to SP-A(-/-) controls, but both were markedly elevated in the SP-A(-/-,DeltaG8-P80) mice. There was no defect in the surface tension-lowering activity of surfactant from SP-A(+/+,DeltaG8-P80) mice, indicating that the inhibitory effect of DeltaG8-P80 on surface activity can be overcome by wild type levels of mouse SP-A. The surface activity of surfactant isolated from the SP-A(-/-,rSP-A) but not the SP-A(-/-,DeltaG8-P80) mice was more resistant than SP-A(-/-) littermate control animals to inhibition by serum proteins in vitro. Pressure volume relationships of lungs from the SP-A(-/-), SP-A(-/-,rSP-A), and SP-A(-/-,DeltaG8-P80) lines were very similar. These data indicate that expression of SP-A in the pulmonary epithelium of SP-A(-/-) animals restores tubular myelin formation and resistance of isolated surfactant to protein inhibition by a mechanism that is dependent on the collagen-like region.

Surfactant protein deficiency in familial interstitial lung disease.

OBJECTIVE: To determine the contribution of surfactant protein abnormalities to the development of chronic lung injury in a familial form of interstitial lung disease. STUDY DESIGN: An 11-year-old girl, her sister, and their mother who were diagnosed with chronic interstitial lung disease underwent laboratory investigation of surfactant protein expression in bronchoalveolar lavage fluid and lung biopsy specimens. Nineteen patients with idiopathic pulmonary fibrosis and 9 patients who were investigated for pulmonary malignancy but who did not have interstitial lung disease served as control subjects. RESULTS: The 3 family members were found to have absent surfactant protein C (SP-C) and decreased levels of SP-A and SP-B in bronchoalveolar lavage fluid (BALF). Immunostaining for pulmonary surfactant proteins in lung biopsy specimens obtained from both children demonstrated a marked decrease of pro-SP-C in the alveolar epithelial cells but strong staining for pro-SP-B, SP-B, SP-A, and SP-D. No deviations from published surfactant protein B or C coding sequences were identified by DNA sequence analysis. All control subjects had a detectable level of SP-C in the BALF. CONCLUSION: The apparent absence of SP-C and a decrease in the levels of SP-A and SP-B are associated with familial interstitial lung disease.

Expression of thyroid transcription factor-1, surfactant proteins, type I cell-associated antigen, and Clara cell secretory protein in pulmonary hypoplasia.

Pulmonary hypoplasia (PH) is a developmental abnormality characterized by diminished distal lung parenchyma. Recent studies have demonstrated that thyroid transcription factor 1 (TTF-1), a member of NKx2 family of homeodomain transcription factors, plays an important role in lung organogenesis and lung epithelial gene expression. In order to evaluate whether abnormal expression of TTF-1 contributes to the pathophysiology of PH, we studied the expression of TTF-1, as well as that of the surfactant proteins (SPs), Clara cell secretory protein (CCSP), and type I cell-associated antigen (T1 cell-Ag), in PH. Immunolocalization patterns of these proteins were evaluated in 15 cases of PH with different associated diseases and compared with those of 14 matched controls. Our study demonstrated that the concentration gradient of TTF-1 along the proximal-distal axis in normal fetal lung is disrupted in PH after 24 weeks gestational age, while the expression of the SPs, CCSP, and T1 cell-Ag seemed to be preserved. We conclude that a normal TTF-1 expression pattern might be crucial in the control of distal lung development. Failure to switch off expression of TTF-1 in PH of more than 24 weeks gestational age may be a final common pathway leading to PH associated with the disease processes investigated in this study.

TGF-beta1 perturbs vascular development and inhibits epithelial differentiation in fetal lung in vivo.

Members of the transforming growth factor beta (TGF-beta) family of polypeptides have been implicated in morphogenesis and differentiation in numerous tissues, including the lung. In order to further define effects of TGF-beta signaling in lung morphogenesis, a constitutively active form of TGF-beta1 was expressed in respiratory epithelial cells of the fetal mouse lung in vivo. Expression of TGF-beta1 arrested lung morphogenesis in the pseudoglandular stage of development, inhibiting synthesis of differentiation-dependent proteins, SP-B, SP-C, and CCSP, and maintaining embryonic patterns of staining for thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3beta (HNF-3beta). The pulmonary mesenchyme was thickened and vascular density was increased by TGF-beta1. TGF-beta1 decreased expression of vascular endothelial growth factor-A (VEGF-A) mRNA and protein, and the abundance of Flk-1 mRNA in the lung mesenchyme. Distribution of platelet-endothelial cell adhesion molecule (PECAM)-1, a marker of pulmonary blood vessels, was altered, and ultrastructural studies demonstrated that TGF-beta1 inhibited vascular development in the fetal lung. TGF-beta1 perturbed both epithelial cell differentiation and formation of the pulmonary vasculature, supporting the concept that precise control of signaling via the TGF-beta receptor pathway is critical for normal lung morphogenesis.

Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the Forkhead Box f1 transcription factor.

Decreased pulmonary expression of Forkhead Box f1 (Foxf1) transcription factor was associated with lethal alveolar hemorrhage in 55% of the Foxf1 +/- newborn mice. The severity of the pulmonary abnormalities correlates with the levels of Foxf1 mRNA. Defects in alveolarization and vasculogenesis were observed in subsets of the Foxf1 +/- mice with relatively low levels of expression from the normal Foxf1 allele. Lung hemorrhage was coincident with disruption of the mesenchymal-epithelial cell interfaces in the alveolar and bronchiolar regions of the lung parenchyma and was associated with increased apoptosis and reduced surfactant protein B (SP-B) expression. Finally, the lung defect associated with the Foxf1 +/- mutation was accompanied by reduced expression of vascular endothelial growth factor (VEGF), the VEGF receptor 2 (Flk-1), bone morphogenetic protein 4 (Bmp-4), and the transcription factors of the Brachyury T-Box family (Tbx2-Tbx5) and Lung Kruppel-like Factor. Reduction in the level of Foxf1 caused neonatal pulmonary hemorrhage and abnormalities in alveologenesis, implicating this transcription factor in the regulation of mesenchyme-epithelial interaction critical for lung morphogenesis.