Concentrated Secretome of Adipose Stromal Cells Limits Influenza A Virus-Induced Lung Injury in Mice.

Despite vaccination and antivirals, influenza remains a communicable disease of high burden, with limited therapeutic options available to patients that develop complications. Here, we report the development and preclinical characterization of Adipose Stromal Cell (ASC) concentrated secretome (CS), generated by process adaptable to current Good Manufacturing Practices (cGMP) standards. We demonstrate that ASC-CS limits pulmonary histopathological changes, infiltration of inflammatory cells, protein leak, water accumulation, and arterial oxygen saturation (spO2) reduction in murine model of lung infection with influenza A virus (IAV) when first administered six days post-infection. The ability to limit lung injury is sustained in ASC-CS preparations stored at -80 °C for three years. Priming of the ASC with inflammatory factors TNFα and IFNγ enhances ASC-CS ability to suppress lung injury. IAV infection is associated with dramatic increases in programmed cell death ligand (PDL1) and angiopoietin 2 (Angpt2) levels. ASC-CS application significantly reduces both PDL1 and Angpt2 levels. Neutralization of PDL1 with anti-mouse PDL1 antibody starting Day6 onward effectively ablates lung PDL1, but only non-significantly reduces Angpt2 release. Most importantly, late-phase PDL1 neutralization results in negligible suppression of protein leakage and inflammatory cell infiltration, suggesting that suppression of PDL1 does not play a critical role in ASC-CS therapeutic effects.

Distinct Factors Secreted by Adipose Stromal Cells Protect the Endothelium From Barrier Dysfunction and Apoptosis.

We have shown previously that adipose stromal cell (ASC)-derived conditioned media (CM) limited lung injury, endothelial barrier dysfunction, and apoptosis. Here, we used endothelial hyperpermeability and apoptosis assays to investigate how concentration processes affect endothelium-directed bioactivity of ASC-CM and to gain information on the nature of bioactive factors. Comparison of ASC-CM concentrated with differential molecular weight (MW) cutoff filters showed that endothelial barrier protection depended on the species-specific factors in ASC-CM fractionated with MW > 50 kDa. Known barrier regulators-keratin growth factor (KGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF)-were detected in ASC-CM fraction of > 100 kDa. Pretreatment of endothelial monolayers with concentrations of KGF, VEGF, and HGF detected in ASC-CM showed that only KGF and HGF protect the endothelium from barrier dysfunction. Depletion of KGF and HGF from ASC-CM attenuated ASC-CM's ability to protect the endothelial barrier. In contrast to barrier-protective factors, apoptosis-protective factors fractionated with MW < 3 kDa and were not species-specific. Application of donors of apoptosis-mitigating gases showed that the CO donor carbon monoxide-releasing molecule 2 (CORM2) protected the endothelium from apoptosis, while the H2S donor NaSH did not. Knockdown of CO-generating heme oxygenase 1 in ASC attenuated ASC-CM's ability to protect the endothelium from apoptosis. We have shown that tumor necrosis factor alpha (TNFα)-induced apoptosis in endothelium is c-Jun N-terminal kinase (JNK)-dependent, and JNK activation is inhibited by ASC-CM pretreatment of endothelial cells. ASC-CM from heme oxygenase 1-depleted ASC displayed attenuated ability to suppress endothelial JNK activation, suggesting that CO-mediated protection of the endothelium from apoptosis is achieved by the downregulation of the JNK pathway. Altogether, our results demonstrate that the concentration of ASC-CM with low MW cutoff filters significantly reduces its anti-apoptotic activity while preserving its barrier-protective activity.

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death.

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury.

Influenza A virus (IAV) remains a major worldwide health threat, especially to high-risk populations, including the young and elderly. There is an unmet clinical need for therapy that will protect the lungs from damage caused by lower respiratory infection. Here, we analyzed the role of EMAPII, a stress- and virus-induced pro-inflammatory and pro-apoptotic factor, in IAV-induced lung injury. First, we demonstrated that IAV induces EMAPII surface translocation, release, and apoptosis in cultured endothelial and epithelial cells. Next, we showed that IAV induces EMAPII surface translocation and release to bronchoalveolar lavage fluid (BALF) in mouse lungs, concomitant with increases in caspase 3 activity. Injection of monoclonal antibody (mAb) against EMAPII attenuated IAV-induced EMAPII levels, weight loss, reduction of blood oxygenation, lung edema, and increase of the pro-inflammatory cytokine TNF alpha. In accordance with the pro-apoptotic properties of EMAPII, levels of caspase 3 activity in BALF were also decreased by mAb treatment. Moreover, we detected EMAPII mAb-induced increase in lung levels of M2-like macrophage markers YM1 and CD206. All together, these data strongly suggest that EMAPII mAb ameliorates IAV-induced lung injury by limiting lung cell apoptosis and shifting the host inflammatory setting toward resolution of inflammation.

AMD3100 ameliorates cigarette smoke-induced emphysema-like manifestations in mice.

We have shown that cigarette smoke (CS)-induced pulmonary emphysema-like manifestations are preceded by marked suppression of the number and function of bone marrow hematopoietic progenitor cells (HPCs). To investigate whether a limited availability of HPCs may contribute to CS-induced lung injury, we used a Food and Drug Administration-approved antagonist of the interactions of stromal cell-derived factor 1 (SDF-1) with its chemokine receptor CXCR4 to promote intermittent HPC mobilization and tested its ability to limit emphysema-like injury following chronic CS. We administered AMD3100 (5mg/kg) to mice during a chronic CS exposure protocol of up to 24 wk. AMD3100 treatment did not affect either lung SDF-1 levels, which were reduced by CS, or lung inflammatory cell counts. However, AMD3100 markedly improved CS-induced bone marrow HPC suppression and significantly ameliorated emphysema-like end points, such as alveolar airspace size, lung volumes, and lung static compliance. These results suggest that antagonism of SDF-1 binding to CXCR4 is associated with protection of both bone marrow and lungs during chronic CS exposure, thus encouraging future studies of potential therapeutic benefit of AMD3100 in emphysema.

Pulmonary Retention of Adipose Stromal Cells Following Intravenous Delivery Is Markedly Altered in the Presence of ARDS.

Transplantation of mesenchymal stromal cells (MSCs) has been shown to effectively prevent lung injury in several preclinical models of acute respiratory distress syndrome (ARDS). Since MSC therapy is tested in clinical trials for ARDS, there is an increased need to define the dynamics of cell trafficking and organ-specific accumulation. We examined how the presence of ARDS changes retention and organ-specific distribution of intravenously delivered MSCs isolated from subcutaneous adipose tissue [adipose-derived stem cells (ADSCs)]. This type of cell therapy was previously shown to ameliorate ARDS pathology. ARDS was triggered by lipopolysaccharide (LPS) aspiration, 4 h after which 300,000 murine CRE+ ADSCs were delivered intravenously. The distribution of ADSCs in the lungs and other organs was assessed by real-time polymerase chain reaction (PCR) of genomic DNA. As anticipated, the majority of delivered ADSCs accumulated in the lungs of both control and LPS-challenged mice, with minor amounts distributed to the liver, kidney, spleen, heart, and brain. Interestingly, within 2 h following ADSC administration, LPS-challenged lungs retained significantly lower levels of ADSCs compared to control lungs (∼7% vs. 15% of the original dose, respectively), whereas the liver, kidney, spleen, and brain of ARDS-affected animals retained significantly higher numbers of ADSCs compared to control animals. In contrast, 48 h later, only LPS-challenged lungs continued to retain ADSCs (∼3% of the original dose), whereas the lungs of control animals and nonpulmonary organs in either control or ARDS mice had no detectable levels of ADSCs. Our data suggest that the pulmonary microenvironment during ARDS may lessen the pulmonary capillary occlusion by MSCs immediately following cell delivery while facilitating pulmonary retention of the cells.

Conditioned media from adipose stromal cells limit lipopolysaccharide-induced lung injury, endothelial hyperpermeability and apoptosis.

BACKGROUND: Acute Respiratory Distress Syndrome (ARDS) is a condition that contributes to morbidity and mortality of critically ill patients. We investigated whether factors secreted by adipose stromal cells (ASC) into conditioned media (ASC-CM) will effectively decrease lung injury in the model of lipopolysaccharide (LPS)-induced ARDS. METHODS: To assess the effect of ASC-CM on ARDS indices, intravenous delivery of ASC and ASC-CM to C57Bl/6 mice was carried out 4 h after LPS oropharyngeal aspiration; Evans Blue Dye (EBD) was injected intravenously 1 h prior to animal sacrifice (48 h post-LPS). Lungs were either fixed for histopathology, or used to extract bronchoalveolar lavage fluid (BALF) or EBD. To assess the effect of ASC-CM on endothelial barrier function and apoptosis, human pulmonary artery endothelial cells were treated with ASC-CM for 48-72 h. RESULTS: ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6. White Blood Cells (WBC) from BALF of LPS-challenged mice receiving ASC-CM had decreased reactive oxygen species (ROS) generation compared to WBC from LPS-challenged mice receiving control media injection. Treatment of pulmonary endothelial monolayers with ASC-CM significantly suppressed H2O2-induced leakage of FITC dextran and changes in transendothelial resistance, as well as gap formation in endothelial monolayer. ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3. ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation. CONCLUSIONS: Factors secreted by ASC efficiently reduce ARDS indices, endothelial barrier hyperpermeability, and activation of pro-inflammatory and pro-apoptotic pathways in endothelium.

Dimethylarginine dimethylaminohydrolase II overexpression attenuates LPS-mediated lung leak in acute lung injury.

Acute lung injury (ALI) is a severe hypoxemic respiratory insufficiency associated with lung leak, diffuse alveolar damage, inflammation, and loss of lung function. Decreased dimethylaminohydrolase (DDAH) activity and increases in asymmetric dimethylarginine (ADMA), together with exaggerated oxidative/nitrative stress, contributes to the development of ALI in mice exposed to LPS. Whether restoring DDAH function and suppressing ADMA levels can effectively ameliorate vascular hyperpermeability and lung injury in ALI is unknown, and was the focus of this study. In human lung microvascular endothelial cells, DDAH II overexpression prevented the LPS-dependent increase in ADMA, superoxide, peroxynitrite, and protein nitration. DDAH II also attenuated the endothelial barrier disruption associated with LPS exposure. Similarly, in vivo, we demonstrated that the targeted overexpression of DDAH II in the pulmonary vasculature significantly inhibited the accumulation of ADMA and the subsequent increase in oxidative/nitrative stress in the lungs of mice exposed to LPS. In addition, augmenting pulmonary DDAH II activity before LPS exposure reduced lung vascular leak and lung injury and restored lung function when DDAH activity was increased after injury. Together, these data suggest that enhancing DDAH II activity may prove a useful adjuvant therapy to treat patients with ALI.

Neutral sphingomyelinase 2 deficiency is associated with lung anomalies similar to emphysema.

Neutral sphingomyelinase 2 (nSMase2) upregulation was recently demonstrated to serve as a molecular link between smoke inhalation and emphysematous changes in lungs. Here we report that nSMase2 deficit impairs lung development in mice. We have shown previously that fragilitas ossium (fro) mice carry a mutation in the Smpd3 gene, rendering nSMase2 catalytically inactive. Analysis of lung phenotype revealed that fro mice have abnormally enlarged alveoli and increased compliance of the respiratory system, similar to morphological and functional manifestations of emphysema. Analysis of sphingolipid content in fro lungs revealed a decreased level of C14:0 ceramide but no significant alterations in the levels of sphingosine or sphingosine-1-phosphate. Altogether, our data suggest that nSMase2 activity and ceramide level are critical for lung development and function. Based on our data, ceramide can no longer be viewed as a lipid solely detrimental to lung function.

Taxol alleviates 2-methoxyestradiol-induced endothelial permeability.

We have previously shown that the anti-cancer agent 2-methoxyestradiol (2ME) induces hyperpermeability across endothelial monolayers. Here, we show that both microtubule disruptor, 2ME, and microtubule stabilizer, paclitaxel (taxol), increase vascular lung permeability in vitro and in vivo. Simultaneous application of 2ME and taxol alleviates 2ME-induced endothelial barrier dysfunction, which is evident by the decreased Evans Blue Dye accumulation in lung tissue and increased transendothelial resistance across monolayers. 2ME significantly increases the level of p38 and MLC phosphorylation in both endothelial monolayers and murine lungs; this increase is suppressed in the presence of taxol. Taxol treatment leads to an immediate and sustained increase in tubulin acetylation in human pulmonary artery endothelial cells (HPAEC). Surprisingly, 2ME treatment also increases tubulin acetylation; however, the onset of this process is delayed and coincides with the stage of a partial barrier restoration in HPAEC monolayer. Inhibition of histone deacetylase 6 (HDAC6) with tubacin increases tubulin acetylation level, suppresses 2ME-induced HSP27 and MLC phosphorylation, and decreases 2ME-induced barrier dysfunction, suggesting barrier-protective and/or barrier-restorative role for tubulin acetylation in vascular endothelium.

TIMAP protects endothelial barrier from LPS-induced vascular leakage and is down-regulated by LPS.

TIMAP is a regulatory subunit of protein phosphatase 1, whose role remains largely unknown. Our recent data suggested that TIMAP is involved in the regulation of barrier function in cultured pulmonary endothelial monolayers [Csortos et al., 2008. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L440-L450]. Here we showed that TIMAP depletion exacerbates lipopolysaccharide (LPS)-induced vascular leakage in murine lung, suggesting that TIMAP has a barrier-protective role in vivo. Real-Time RT PCR analysis revealed that treatment with LPS significantly suppressed Timap mRNA level. This suppression was not achieved via the down-regulation of Timap promoter activity, suggesting that LPS decreased Timap mRNA stability. Pretreatment with protein kinase A (PKA) inhibitor H-89 reduced TIMAP mRNA level, whereas pretreatment with PKA activator, bnz-cAMP, increased this level and attenuated LPS-induced decrease in TIMAP mRNA. Altogether, these data confirmed the barrier-protective role of TIMAP and suggested that barrier-disruptive and barrier-protective agents may employ modulation of TIMAP expression as a mechanism affecting barrier permeability.

Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability.

We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses that ERM proteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylation-deficient ERM mutants, exhibit less attenuation of 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubule-disrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.

Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability.

Up to date, the nature of the sepsis-induced vascular leakage is understood only partially, which limits pharmacological approaches for its management. Here we studied the protective effect of cAMP using endotoxin-induced hyperpermeability as a model for barrier dysfunction observed in gram-negative sepsis. We demonstrated that the alleviation of lipopolysaccharide (LPS)-induced barrier compromise could be achieved by the specific activation of either protein kinase A (PKA) or Epac with cAMP analogs Bnz-cAMP or O-Me-cAMP, respectively. We next studied the involvement of PKA substrates VASP and filamin1 in barrier maintenance and LPS-induced barrier compromise. Depletion of both VASP and filamin1 with the specific siRNAs significantly exacerbated both the quiescent cells barrier and LPS-induced barrier dysfunction, suggesting barrier-protective role of these proteins. VASP depletion was associated with the more severe loss of ZO-1 peripheral staining in response to LPS, whereas filamin1-depleted cells reacted to LPS with more robust stress fiber induction and more profound changes in ZO-1 and VE-cadherin peripheral organization. Both VASP and filamin1 phosphorylation was significantly increased as a result of PKA activation. We next analyzed the effect of VASP and filamin1 depletion on the PKA-dependent alleviation of LPS-induced barrier compromise. We observed that Bnz-cAMP ability to counteract LPS-induced hyperpermeability was attenuated only by VASP, but not filamin1 depletion. Our data indicate that while PKA-dependent VASP phosphorylation contributes to the protective effect of cAMP elicited on LPS-compromised monolayers, filamin1 phosphorylation is unlikely to play a significant role in this process.

Reprint of "The role of cytoskeleton in the regulation of vascular endothelial barrier function" [Microvascular Research 76 (2008) 202-207].

The cytoskeleton is vital to the function of virtually all cell types in the organism as it is required for cell division, cell motility, endo- or exocytosis and the maintenance of cell shape. Endothelial cells, lining the inner surface of the blood vessels, exploit cytoskeletal elements to ensure the integrity of cell monolayer in quiescent endothelium, and to enable the disintegration of the formed barrier in response to various agonists. Vascular permeability is defined by the combination of transcellular and paracellular pathways, with the latter being a major contributor to the inflammation-induced barrier dysfunction. This review will analyze the cytoskeletal elements, which reorganization affects endothelial permeability, and emphasize signaling mechanisms with barrier-protective or barrier-disruptive potential.

The role of cytoskeleton in the regulation of vascular endothelial barrier function.

The cytoskeleton is vital to the function of virtually all cell types in the organism as it is required for cell division, cell motility, endo- or exocytosis and the maintenance of cell shape. Endothelial cells, lining the inner surface of the blood vessels, exploit cytoskeletal elements to ensure the integrity of cell monolayer in quiescent endothelium, and to enable the disintegration of the formed barrier in response to various agonists. Vascular permeability is defined by the combination of transcellular and paracellular pathways, with the latter being a major contributor to the inflammation-induced barrier dysfunction. This review will analyze the cytoskeletal elements, which reorganization affects endothelial permeability, and emphasize signaling mechanisms with barrier-protective or barrier-disruptive potential.

TIMAP is a positive regulator of pulmonary endothelial barrier function.

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

Developmental expression and gene regulation of insulin-like 3 receptor RXFP2 in mouse male reproductive organs.

The mutations of testicular insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. Here we have examined Rxfp2 gene expression at different stages of embryonic and postnatal mouse development in male reproductive tissues employing quantitative RT-PCR and several RXFP2-specific antibodies directed toward different parts of the RXFP2 protein. Receptor expression was markedly increased after birth and was readily detectable in the epididymis, Leydig cells, and germ cells of the testis. The strongest expression was detected in adult mouse cremaster muscle. INSL3 treatment increased cell proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine signaling in these cells and identifying TM3 as a novel in vitro model to study the effects of RXFP2 signaling. We generated Tg(Rxfp2-cre)Aia (Rxfp2-iCre) transgenic mice expressing improved Cre recombinase (iCre) under the control of the 2.4-kb mouse Rxfp2 promoter. The iCre was expressed in the gubernacular ligament at E14.5, indicating that this promoter is able to drive Rxfp2 gene expression during transabdominal testis descent. We demonstrated that the transcription factor Sox9, a known male sex determination factor, is expressed in mouse embryonic gubernacula and upregulated human, but not mouse, promoter luciferase reporter constructs. In conclusion, we have determined the developmental expression profile of INSL3 receptor employing newly characterized RXFP2 antisera and a novel Rxfp2-iCre transgenic mouse model. We determined the promoter region capable of providing the gubernacular-specific expression of Rxfp2. Analysis of RXFP2 promoter identified SOX9 as a new transcriptional enhancer of human gene expression.

Novel mutations involving the INSL3 gene associated with cryptorchidism.

PURPOSE: Cryptorchidism affects 1% to 9% of full-term male neonates. Hypospadias is the second most frequent congenital anomaly seen in newborn males. These pathological conditions are part of the testicular dysgenesis syndrome. Insulin-like factor 3 and LGR8 (leucine-rich repeat-containing G protein-coupled receptor 8), acting as a hormone and a receptor, respectively, are involved in control of the first phase of testicular descent via gubernacular development. MATERIALS AND METHODS: The study group consisted of 184 patients, of whom 52 presented with unilateral cryptorchidism, 37 presented with bilateral cryptorchidism, 19 presented with cryptorchidism and hypospadias, 1 presented with bilateral cryptorchidism and micropenis, and 75 presented with isolated hypospadias. A control panel consisted of 270 controls, including 127 fertile, and 143 fertile noncryptorchid males. Insulin-like factor 3 mutations were analyzed by direct sequencing and restriction enzyme digestion. We analyzed the ability of the mutant insulin-like factor 3 peptides identified in this study to activate LGR8 receptor in an ex vivo assays. RESULTS: We identified 3 novel insulin-like factor 3 variants, including C-19G, V18M and R105H, in 3 of the 109 patients (2.75%) but in none of the 270 controls. The V18M mutation in the insulin-like factor 3 signal peptide had a significant deleterious effect in activating LGR8 receptor in ex vivo studies (p<0.05). To our knowledge we report the first variant in the promoter region of the insulin-like factor 3 gene in a patient with cryptorchidism in association with micropenis. CONCLUSIONS: Mutations involving the insulin-like factor 3 gene may contribute to other anomalies of male genital development, such as micropenis.

Relaxin promotes prostate cancer progression.

PURPOSE: To understand the role of relaxin peptide in prostate cancer, we analyzed the expression of relaxin and its receptor in human prostate cancer samples, the effects of relaxin signaling on cancer cell phenotype in vitro, and the effects of increased serum relaxin concentrations on cancer progression in vivo. EXPERIMENTAL DESIGN: The relaxin and its receptor leucine-rich repeat containing G protein-coupled receptor 7 (LGR7) expression were studied by quantitative reverse transcription-PCR (11 benign and 44 cancer tissue samples) and by relaxin immunohistochemistry using tissue microarrays containing 10 normal and 69 cancer samples. The effects of relaxin treatment and endogenous relaxin/LGR7 suppression via short interfering RNA in PC-3 and LNCaP cells were analyzed in vitro. The effect of transgenic relaxin overexpression [Tg(Rln1)] on cancer growth and survival was evaluated in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP). RESULTS: The relaxin mRNA expression was significantly higher in recurrent prostate cancer samples. In tissue microarrays of the 10 normal tissues, 8 had low staining in epithelial cells, whereas only 1 of 9 high-grade prostatic intraepithelial neoplasia lesions had low expression (P = 0.005) and only 29 of 65 cancers had low expression (P = 0.047). Stimulation with relaxin increased cell proliferation, invasiveness, and adhesion in vitro. The suppression of relaxin/LGR7 via short interfering RNAs decreased cell invasiveness by 90% to 95% and growth by 10% to 25% and increased cell apoptosis 0.6 to 2.2 times. The Tg(Rln1) TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, compared with non-Tg(Rln1) TRAMP animals. CONCLUSIONS: Relaxin signaling plays a role in prostate cancer progression.