Safety and Efficacy of Combined Resveratrol and Sirolimus in Lymphangioleiomyomatosis.

BACKGROUND: A critical need exists to develop remission-inducing therapies for lymphangioleiomyomatosis. RESEARCH QUESTION: Is the addition of resveratrol safe and more efficacious than sirolimus alone in patients with lymphangioleiomyomatosis? STUDY DESIGN AND METHODS: We conducted a phase 2, dose-escalating, open-label trial of resveratrol in patients with lymphangioleiomyomatosis receiving a stable regimen of sirolimus. Resveratrol was started at 250 mg/d and escalated every 8 weeks to maximum dose of 1,000 mg/d over 24 weeks. The primary outcome was ≥ 42% decline in serum vascular endothelial growth factor D (VEGF-D) levels on combined therapy compared with baseline VEGF-D levels on sirolimus. Secondary objectives included an assessment of the safety profile and the effect on lung function and health-related quality of life (HRQOL). Longitudinal change in outcome measures was assessed using linear mixed models. Adverse effects were tabulated using the National Cancer Institute's Common Terminology Criteria for Adverse Events version 4. RESULTS: Twenty-five patients with lymphangioleiomyomatosis with a median age of 51 years were enrolled. Pulmonary function parameters at study inclusion were: FEV1: median absolute, 1.72 L; 64% predicted; FVC: median absolute, 2.99 L; 96% predicted; and diffusing capacity of the lungs for carbon monoxide: median absolute, 14.68 mL/mm Hg/min; 37% predicted. The median serum VEGF-D value at baseline was 617 pg/mL. Patients entered the study with a median sirolimus dose of 2 mg/d with median trough level of 6.3 ng/mL. Despite some GI side effects, the addition of resveratrol was well tolerated. Although the primary outcome was not met, a statistically significant reduction in serum VEGF-D levels and improvement in HRQOL during the study was found. INTERPRETATION: The addition of resveratrol was safe and well tolerated in patients with lymphangioleiomyomatosis taking sirolimus and was associated with modest improvement in HRQOL. Larger controlled trials of this combination might be warranted to assess definitively the usefulness of resveratrol as an additive therapy in lymphangioleiomyomatosis. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03253913; URL: www. CLINICALTRIALS: gov.

Vasohibin 2 promotes lymphangiogenesis of lung squamous cell carcinoma through snail-dependent vascular endothelial growth factor-D (VEGF-D) signaling pathway.

Background: Tumor lymphatic metastasis is mostly dependent on lymphangiogenesis, which was less studied compared to angiogenesis and the molecular mechanisms involved remained unclear. Methods: We analyzed the mRNA expression profiles of 937 primary lung squamous cell carcinoma (LUSC) samples from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to explore the genes related to lymphatic metastasis in LUSC. We focused on vasohibin 2 (VASH2) and investigated its biological functions in LUSC proliferation, apoptosis, migration, invasion, as well as lymphangiogenesis capacity by forced over-expressing VASH2 in LUSC cell line H520 in vitro and in vivo. We also evaluated the anti-tumor efficacy of specific anti-VASH2 antibody in LUSC xenograft-bearing mice models. Results: Vasohibin2 (VASH2) was filtered out as a significant predictive factor of poor prognosis and lymphatic metastasis in LUSC patients both in public datasets and an independent Chinese LUSC cohort. VASH2 promoted the proliferation and invasion of LUSC cells in vitro and vivo. Forced over-expression of VASH2 in LUSC cells promoted the amplification and tube formation capacity of human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) via up-regulating vascular endothelial growth factor-D (VEGF-D), which could be reversed via Snail inhibition. Furthermore, blocking VASH2/VEGF-D signaling using specific antibodies dramatically inhibited tumor growth in mice by interfering with the proliferation of cancer cells and lymphangiogenesis in tumor tissues. Conclusions: In conclusion, VASH2 facilitated lymphangiogenesis and tumor growth in a Snail-dependent manner and might serve as a novel biomarker for early diagnosis and prognosis prediction, as well as a potential therapeutic target in LUSC.

Diagnostic performance of VEGF-D for lymphangioleiomyomatosis: a meta-analysis / Desempenho diagnóstico do VEGF-D para linfangioleiomiomatose: meta-análise

ABSTRACT Objective: VEGF-D is a potential biomarker for lymphangioleiomyomatosis (LAM); however, its diagnostic performance has yet to be systematically studied. Methods: We searched PubMed, EMBASE, Scopus, Web of Science, and Cochrane Library to identify primary studies on VEGF-D in relation to the diagnosis of LAM. The quality of the studies was evaluated using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). Summary estimates of diagnostic accuracy were pooled using a bivariate random effects model. Subgroup and sensitivity analyses were performed to explore possible heterogeneity. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) was applied to rate the quality of evidence and indicate the strength of recommendations. Results: Ten studies involving 945 patients were of high risk in quality, as assessed using the QUADAS-2. The pooled diagnostic parameters were indicated as follows: sensitivity = 0.82 (95% CI, 0.71-0.90); specificity = 0.98 (95% CI, 0.94-0.99); and diagnostic OR = 197 (95% CI, 66-587). The AUC of summary ROC analysis was 0.98. The subgroup and sensitivity analyses revealed that the overall performance was not substantially affected by the composition of the control group, prespecified cutoff value, the country of origin, or different cutoff values (p > 0.05 for all). A strong recommendation for serum VEGF-D determination to aid in the diagnosis of LAM was made according to the GRADE. Conclusions: VEGF-D seems to have great potential implications for the diagnosis of LAM in clinical practice due to its excellent specificity and suboptimal sensitivity.

RESUMO Objetivo: O VEGF-D é um potencial biomarcador para linfangioleiomiomatose (LAM); entretanto, seu desempenho diagnóstico ainda não foi sistematicamente estudado. Métodos: Foram realizadas buscas nos bancos de dados PubMed, EMBASE, Scopus, Web of Science e Cochrane Library para identificar estudos primários sobre o VEGF-D com relação ao diagnóstico de LAM. A qualidade dos estudos foi avaliada por meio da ferramenta Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). As estimativas sumárias de acurácia diagnóstica foram combinadas utilizando um modelo bivariado de efeitos aleatórios. Análises de subgrupo e de sensibilidade foram realizadas para explorar possíveis heterogeneidades. O sistema Grading of Recommendations Assessment, Development, and Evaluation (GRADE) foi aplicado para avaliar a qualidade das evidências e indicar a força das recomendações. Resultados: Dez estudos envolvendo 945 pacientes eram de alto risco em qualidade, segundo a ferramenta QUADAS-2. Os parâmetros diagnósticos combinados foram indicados da seguinte forma: sensibilidade = 0,82 (IC95%: 0,71-0,90); especificidade = 0,98 (IC95%: 0,94-0,99); e OR diagnóstica = 197 (IC95%: 66-587). A ASC da análise summary ROC foi de 0,98. As análises de subgrupo e de sensibilidade revelaram que o desempenho global não foi substancialmente afetado pela composição do grupo controle, valor de corte pré-especificado, país de origem ou diferentes valores de corte (p > 0,05 para todos). Uma forte recomendação para a dosagem de VEGF-D sérico para auxiliar no diagnóstico de LAM foi feita de acordo com o sistema GRADE. Conclusões: O VEGF-D parece ter grandes implicações potenciais para o diagnóstico de LAM na prática clínica em virtude da excelente especificidade e sensibilidade subótima.

Reduced risk of recurrent pneumothorax for sirolimus therapy after surgical pleural covering of entire lung in lymphangioleiomyomatosis.

BACKGROUND: Patients with lymphangioleiomyomatosis (LAM) frequently experience pneumothorax. Although sirolimus is the standard therapy for LAM, its effect on pneumothorax is controversial. Recently, total pleural covering (TPC) and modified TPC (mTPC) were introduced as surgical treatment options for pneumothorax for patients with LAM. However, the effect of sirolimus on the recurrence of pneumothorax in patients who underwent the treatments is still uncertain. We hypothesized that some clinical factors including sirolimus treatment could predict postoperative recurrence of pneumothorax. In order to clarify this hypothesis, we retrospectively analyzed the clinical data from 18 consecutive patients with LAM who underwent 24 surgical pleural covering of entire lung (SPC) as 17 TPC and 7 mTPC against pneumothoraces from surgical database between January 2005 and January 2019, and we determined the predictors of postoperative recurrence. RESULTS: Of the 24 surgeries of SPC, 14 surgeries (58.3%) had a history of two or more ipsilateral pneumothoraces, and 11 surgeries (45.8%) had a history of ipsilateral pleural procedures before SPC. Sixteen surgeries (66.6%) in 12 patients received treatment of sirolimus after SPC (sirolimus group). With a median follow-up time of 69.0 months after SPC, four surgeries (16.6%) in three patients had a postoperative recurrence, and the 5-year recurrence-free survival (RFS) after SPC was 82.9%. In patients with postoperative recurrence, serum level of vascular endothelial growth factors D was significantly higher than that in those with non-recurrence (3260.5 vs. 892.7 pg/mL, p = 0.02), and the rate of sirolimus treatment in the recurrence group was significantly lower than that in the no-recurrence group (0 vs. 80%, p = 0.006). The log-rank test showed that the RFS of the sirolimus group (sirolimus use after SPC) was significantly better than that of the non-sirolimus group (p = 0.001), and no significant difference was observed for other factors. CONCLUSION: We first reported sirolimus might effectively suppress the recurrence of pneumothoraces in LAM patients who received SPC. Sirolimus induction after SPC (TPC or mTPC) might be a feasible option for frequent pneumothorax in LAM.

Clinical development and evaluation of a VEGF-D assay in plasma from patients with metastatic colorectal cancer in the RAISE study.

BACKGROUND: Vascular endothelial growth factor (VEGF)-D was identified as a potential predictive biomarker for ramucirumab efficacy in second-line metastatic colorectal cancer using a research use only (RUO) assay. We describe results with a new assay for detecting VEGF-D in human plasma. METHODS: In RAISE (Clinical Trial Registration: NCT01183780), 1072 patients were randomized 1:1 to ramucirumab or placebo plus FOLFIRI. All patients were then randomized 1:2 to marker exploratory (ME) and marker confirmatory (MC) groups, and those with plasma samples were analyzed accordingly. A new assay validated for investigational use only (IUO) was used to measure VEGF-D levels in plasma, which were analyzed for correlation with overall and progression-free survival (OS/PFS). IUO assay data were compared with historical RUO assay data. RESULTS: ME subset analyses determined the optimal cutpoint of 5.4 ng/mL for defining high/low VEGF-D subgroups. In the combined ME/MC placebo arms, OS/PFS were numerically greater for patients with low vs high VEGF-D (OS: 12.8 vs 11.1 months; PFS: 5.6 vs 4.2 months). In patients with high VEGF-D, ramucirumab vs placebo demonstrated a numerically greater improvement in OS and PFS. Differential efficacy by VEGF-D level was statistically significant for PFS, but not OS. CONCLUSION: In patients with high VEGF-D, ramucirumab demonstrated a greater improvement in OS and PFS vs placebo; however, baseline VEGF-D level was not predictive of ramucirumab OS benefit using VEGF-D assay for IUO. The RAISE intent-to-treat results remain valid.

Vascular endothelial growth factor D is a biomarker of fluid overload in haemodialysis patients.

BACKGROUND: Improved understanding and assessment of the complex physiology of volume regulation in haemodialysis (HD) patients are required to improve patient care and reduce mortality associated with fluid overload (FO). METHODS: We searched for FO-related biomarkers among 184 peptides associated with cardiovascular disease in a cohort of 30 HD patients. First, we assessed the direct impact of HD on the peptides of interest by comparing plasma concentrations before and after treatment. Then, we compared cardiovascular peptide profiles between patients with and without FO as defined by bioimpedance analysis (BIA). The plasma concentration of selected candidate biomarkers for FO was determined by enzyme-linked immunosorbent assay (ELISA) and correlated with previously described FO-related clinical and laboratory parameters. For validation, results were confirmed in an independent cohort of 144 HD patients. RESULTS: We found seven peptides positively [NT-proBNP, B-type natriuretic peptide (BNP), vascular endothelial growth factor D (VEGFD), tumour necrosis factor-related apoptosis-inducing ligand receptor 2, growth differentiation factor 15, tumour necrosis factor ligand superfamily member 13B, chitinase-3-like protein 1] and five negatively (leptin, renin, epidermal growth factor receptor, interleukin-1 receptor antagonist, myeloblastin) correlated to FO. In addition to natriuretic peptides, VEGFD emerged as third peptide highly correlated with BIA (ρ = 0.619, P < 0.0001). In line with this, VEGFD concentration verified by ELISA correlated with BIA, BNP and soluble CD146 but not with vascular endothelial growth factor C (VEGFC). Notably, levels of VEGFD were unrelated to cardiac systolic function (P = 0.63), contrary to BNP (P = 0.0003). Finally, we observed that 1-year all-cause mortality was higher in patients with high BNP (P = 0.0002), FO (defined by BIA, P = 0.04) and high VEGFD (P = 0.02), but not with high VEGFC (P = 0.48). CONCLUSION: VEGFD is a novel FO-related biomarker with unique diagnostic and prognostic properties.

Overexpression of VEGF165 is associated with poor prognosis of cervical cancer.

BACKGROUND: Cervical cancer is a major health hazard to Indian women. Human papillomavirus (HPV) infection is an established risk factor for cervical carcinogenesis. However, understanding the cervical cancer biology beyond HPV infection is very crucial to predict aggressive behavior, prognosis, treatment response and survival. In the present study, we explored the role of vascular endothelial growth factor A (VEGFA) isoforms, VEGFC and VEGFD in cervical cancer progression and its association with HPV 16 and 18 infections. MATERIAL AND METHODS: A total of 110 cervical cancer tissues and 50 normal cervical tissues were collected for the study. Reverse transcription-polymerase chain reaction was employed to analyze tissue VEGFA isoforms, VEGFC and VEGFD expression. RESULTS: VEGF165 was significantly higher, whereas VEGFC and VEGFD were significantly lower in malignant cervical carcinoma tissues as compared to normal cervix tissues. Expression levels of VEGF121 and VEGFC were significantly associated with type of tumor growth while VEGF165 was significantly associated with lymph node metastasis. VEGF165 transcript levels were significantly higher in patients with squamous cell carcinoma (SCC) and developed recurrence. Most strikingly, higher VEGF165 expression was significantly associated with worst disease-free survival (DFS) specifically in patients with SCC. CONCLUSION: Association of VEGF165 with lymph node metastasis, disease recurrence and worst DFS indicated that VEGF165 is an important prognostic factor in cervical carcinogenesis.

A woman with dyspnea and recurrent pneumothorax: when dyspnea is not asthma.

Lymphangioleiomyomatosis (LAM) is a rare disease characterized by cystic lung lesions, lymphatic abnormalities, and angiomyolipomas. It can take a significant amount of time to diagnose LAM due to the vague symptoms of fatigue, progressive dyspnea, pneumothorax, and pleural effusion. We present a case of a 29-year-old woman with recurrent spontaneous pneumothorax and progressive dyspnea who was initially misdiagnosed with asthma and was later found to have LAM. As with all rare diagnoses, there needs to be a suspicion of the disease in order for a further workup to be initiated. In patients with a compatible High-resolution CT scan of the chest, a high vascular endothelial growth factor-D (VEGF-D) value is diagnostic for LAM, and no other confirmatory test is needed.

Angiogenesis modulatory factors in subjects with chronic ocular complications of Sulfur Mustard exposure: A case-control study.

BACKGROUND: Chronic ocular complications of Sulfur Mustard (SM) exposure leads to severe ocular morbidity during time. The aim of this study was to compare serum levels of Interleukin 17 (IL-17), IL-12, vascular endothelial growth factor (VEGF)-C, VEGF-D and nitric oxide (NO) in SM-exposed patients versus the control group and to measure tear concentration of VEGF-C only in the SM-exposed group. METHODS: In this prospective case control, 128 SM-exposed patients and 31 healthy control subjects were included. In the case group ocular manifestations were classified to three subgroups of mild (19 cases), moderate (31 cases) and severe (78 cases) forms of disease. Serum levels of IL-17, IL-12, NO, VEGF-C and VEGF-D, in all subjects and tear concentration of VEGF-C in SM-exposed group was evaluated. RESULTS: All subjects were male and mean ±â€¯standard deviation (SD) of age in the case and control groups were 44.9 ±â€¯8.8 and 40.9 ±â€¯10.1 years, respectively. Except for significantly lower serum level of IL-17 (p < 0.001) and NO (p = 0.003), other values were not significantly different. The tear concentration of VEGF-C and serum level of IL-12 were not different between subgroups in the SM-exposed group, yet were significantly lower among those with abnormally dilated and tortuous conjunctival vessels and corneal pannus, respectively (p = 0.01, p = 0.015). CONCLUSIONS: Exposure to SM significantly reduced serum level of IL-17 and NO in the delayed phase, yet did not influence VEGF-C; VEGF-D or IL-12.

Experimental and clinicopathological analysis of HOXB9 in gastric cancer.

The association between homeobox (HOX)B9 expression and tumor malignancy was identified recently. It was reported that HOXB9 induced tumor angiogenesis, and associated with poor prognosis in patients with breast and colon cancer. On the other hand, regional lymph nodes are the most common site of tumor spread, and lymph node metastasis is a major prognostic factor in gastric cancer. It was hypothesized that HOXB9 promotes tumor lymphangiogenesis and induces tumor progression, invasion and metastasis in gastric cancer. The aim of the present study was to evaluate the correlation between HOXB9 expression, prognosis and clinicopathologic factors in patients with gastric cancer, and to assess the contribution of HOXB9 expression to tumor cell lymphangiogenesis in vitro. HOXB9 expression was evaluated by immunohistochemistry in resected tumor tissues from 58 patients with gastric cancer, and the association between prognosis and clinicopathologic factors was determined. HOXB9 gene was overexpressed in human gastric cancer TMK-1 cells and the effect of HOXB9 overexpression on the expression of vascular endothelial growth factor (VEGF)-C, VEGF-D and VEGF receptor (R)-3 was determined. It was demonstrated that the depth of tumor invasion, the number of node metastases, lymphatic invasion and vascular invasion were significantly associated with HOXB9 expression. Overall survival was decreased in patients with HOXB9 expression. The mRNA expression of VEGF-D but not of VEGF-C and VEGFR-3 was increased in HOXB9-overexpressing TMK-1 cells compared with control cells. In conclusion, HOXB9 expression was positively correlated with gastric cancer progression and lymphangiogenesis marker expression. HOXB9 may be associated with lymphogenic metastasis.

Viable cryopreserved umbilical tissue (vCUT) reduces post-operative adhesions in a rabbit abdominal adhesion model.

Post-operative adhesions, a common complication of surgery, cause pain, impair organ functionality, and often require additional surgical interventions. Control of inflammation, protection of injured tissue, and rapid tissue repair are critical for adhesion prevention. Adhesion barriers are biomaterials used to prevent adhesions by physical separation of opposing injured tissues. Current adhesion barriers have poor anti-inflammatory and tissue regenerative properties. Umbilical cord tissue (UT), a part of the placenta, is inherently soft, conforming, biocompatible, and biodegradable, with antimicrobial, anti-inflammatory, and antifibrotic properties, making it an attractive alternative to currently available adhesion barriers. While use of fresh tissue is preferable, availability and short storage time limit its clinical use. A viable cryopreserved UT (vCUT) "point of care" allograft has recently become available. vCUT retains the extracellular matrix, growth factors, and native viable cells with the added advantage of a long shelf life at -80 °C. In this study, vCUT's anti-adhesion property was evaluated in a rabbit abdominal adhesion model. The cecum was abraded on two opposing sides, and vCUT was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Gross and histological evaluation was performed at 7, 28, and 67 days post-surgery. No adhesions were detectable on the vCUT treated side at all time points. Histological scores for adhesion, inflammation, and fibrosis were lower on the vCUT treated side as compared to the control side. In conclusion, the data supports the use of vCUT as an adhesion barrier in surgical procedures.

Sonic hedgehog promotes endothelial differentiation of bone marrow mesenchymal stem cells via VEGF-D.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) have been proved to be capable of differentiating into endothelial cells (ECs), however, the differentiation efficiency is rather low. Sonic hedgehog (Shh), an important factor in vascular development and postnatal angiogenesis, exerted promotional effect on new vessel formation in the ischemic animal models. Therefore, the current study aims to investigate whether Shh could induce the endothelial differentiation of BMSCs both in vitro and in vivo, as well as the mechanism of differentiation induction. METHODS: The current study over-expressed Shh in BMSCs by lentivirus transduction. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the angiogenic factors in both control BMSCs and Shh over-expressed BMSCs. Immunocytochemistry was also conducted to examine the EC markers. Angiogenesis was determined by in vitro tube-forming assay on Matrigel and in vivo Matrigel plug in severe combined immunodeficient (SCID) mice. Last, mRNA sequencing analysis was used to elaborate the underlying mechanisms. Loss of function study was performed by vascular endothelial growth factor D (VEGF-D) siRNA. RESULTS: Shh expression was increased by about 3,000-fold and 5,000-fold at 3 days-transfection and 7 days-transfection, respectively. Patched 1 (Ptch1), the receptor for Shh, had a two-fold increase after transduction. The angiogenic factors such as hepatocyte growth factor (HGF), angiopoietin-1 (Ang-1), insulin-like growth factor 1 (IGF1) and vascular endothelial growth factor A (VEGF-A) had at least a 1.5-fold increase after transduction. Expression of EC-lineage markers, CD31 and VE-cadherin, on Shh-overexpressed BMSCs were increasingly detected by immunocytostaining. Angiogenesis of BMSCs could be efficiently induced by Shh overexpression in the in vitro tube-formation assay and in vivo Matrigel plug. Additionally, mRNA sequencing analysis revealed that Shh activation upregulated the expression of several pro-angiogenic factors, like Angptl4, Egfl6, VEGF-D. Loss of function study by VEGF-D siRNA confirmed that Shh enhanced the angiogenic ability of BMSCs via VEGF-D. CONCLUSIONS: This study demonstrated that Shh could promote endothelial differentiation of BMSCs via VEGF-D.

Deletion of Lymphangiogenic and Angiogenic Growth Factor VEGF-D Leads to Severe Hyperlipidemia and Delayed Clearance of Chylomicron Remnants.

Objective- Dyslipidemia is one of the key factors behind coronary heart disease. Blood and lymphatic vessels play pivotal roles in both lipoprotein metabolism and development of atherosclerotic plaques. Recent studies have linked members of VEGF (vascular endothelial growth factor) family to lipid metabolism, but the function of VEGF-D has remained unexplored. Here, we investigated how the deletion of VEGF-D affects lipid and lipoprotein metabolism in atherogenic LDLR-/- ApoB100/100 mice. Approach and Results- Deletion of VEGF-D (VEGF-D-/-LDLR-/-ApoB100/100) led to markedly elevated plasma cholesterol and triglyceride levels without an increase in atherogenesis. Size distribution and hepatic lipid uptake studies confirmed a delayed clearance of large chylomicron remnant particles that cannot easily penetrate through the vascular endothelium. Mechanistically, the inhibition of VEGF-D signaling significantly decreased the hepatic expression of SDC1 (syndecan 1), which is one of the main receptors for chylomicron remnant uptake when LDLR is absent. Immunohistochemical staining confirmed reduced expression of SDC1 in the sinusoidal surface of hepatocytes in VEGF-D deficient mice. Furthermore, hepatic RNA-sequencing revealed that VEGF-D is also an important regulator of genes related to lipid metabolism and inflammation. The lack of VEGF-D signaling via VEGFR3 (VEGF receptor 3) led to lowered expression of genes regulating triglyceride and cholesterol production, as well as downregulation of peroxisomal ß-oxidation pathway. Conclusions- These results demonstrate that VEGF-D, a powerful lymphangiogenic and angiogenic growth factor, is also a major regulator of chylomicron metabolism in mice.

Cystic Lung Disease in Genetic Syndromes with Deficient Tumor Suppressor Gene Function.

Cystic lung diseases constitute a distinct group of rare lung disorders, among which two result from monogenic defects affecting tumor suppressor genes: lymphangioleiomyomatosis, either sporadic or associated with tuberous sclerosis complex, and Birt-Hogg-Dubé syndrome. These disorders have similarities in their clinical expression, including occurrence in young adults, multiple pulmonary cysts, recurrent pneumothorax, skin hamartomas, and renal tumors. However, they markedly differ in their gender distribution, pathogenesis, disease course, and prognosis. Our knowledge on these two rare conditions is rapidly expanding. Management of lymphangioleiomyomatosis has substantially improved in the past decade with the understanding of its pathogenic mechanisms, the discovery of an effective therapy, and development of large cohorts and international guidelines. Birt-Hogg-Dubé syndrome has been described more recently and still awaits deeper understanding of its pathophysiology.

A Phase II Clinical Trial of an Aromatase Inhibitor for Postmenopausal Women with Lymphangioleiomyomatosis.

RATIONALE: Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease that predominantly affects women and can worsen with pregnancy, estrogen treatment, and the menstrual cycle, suggesting an important role for estrogen in disease pathogenesis. OBJECTIVES: To assess the efficacy and safety of the aromatase inhibitor letrozole in the treatment of LAM. METHODS: Seventeen postmenopausal women with LAM were enrolled in this phase II trial and randomized to receive letrozole 2.5 mg daily (n = 9) or placebo (n = 8) for a period of 12 months. Five patients in each group were also taking sirolimus at baseline and remained on the drug throughout the treatment period. Lung function, exercise capacity, quality of life, and serum vascular endothelial growth factor D (VEGF-D) were measured at baseline and at 3-month intervals. RESULTS: Fifteen patients completed the study. Two patients withdrew. There were no differences in adverse events in the letrozole and placebo groups. The target enrollment of 25 patients per arm was not met, so the efficacy of letrozole could not be assessed as planned. After adjusting for sirolimus use, we found that the rate of change in FEV1 for all subjects was -3 ± 3 ml/mo (P = 0.4), and for serum VEGF-D, the rate of change was -0.024 ± 0.009 pg/ml/mo (P = 0.015), showing a steeper decline in the letrozole group (-0.029 ± 0.013; P = 0.025). All patients who were taking sirolimus had a reduction in VEGF-D levels from baseline to the last visit, compared with only half of the patients who were not taking sirolimus. In a post hoc analysis, eight matched letrozole-treated-placebo-treated pairs were constructed, six of which demonstrated better FEV1 improvement for the letrozole-treated patients. CONCLUSIONS: Letrozole treatment appears to be safe and well tolerated in postmenopausal patients with LAM, including those taking sirolimus. Enrollment in this trial was compromised by the publication of an effective treatment (sirolimus) in the same month as the study opened, resulting in limited power to detect treatment effects. Post hoc matched pairs exploration studies provide tentative support for additional studies of letrozole in LAM. Considering the reduced rate of lung function decline in postmenopausal patients, future studies will likely require enhanced study designs, such as selective enrollment of those with prognostic biomarkers predictive of decline. Clinical trial registered with www.clinicaltrials.gov (NCT01353209).

Lymphangiogenesis: fuel, smoke, or extinguisher of inflammation's fire?

Lymphangiogenesis is a recognized hallmark of inflammatory processes in tissues and organs as diverse as the skin, heart, bowel, and airways. In clinical and animal models wherein the signaling processes of lymphangiogenesis are manipulated, most studies demonstrate that an expanded lymphatic vasculature is necessary for the resolution of inflammation. The fundamental roles that lymphatics play in fluid clearance and immune cell trafficking from the periphery make these results seemingly obvious as a mechanism of alleviating locally inflamed environments: the lymphatics are simply providing a drain. Depending on the tissue site, lymphangiogenic mechanism, or induction timeframe, however, evidence shows that inflammation-associated lymphangiogenesis (IAL) may worsen the pathology. Recent studies have identified lymphatic endothelial cells themselves to be local regulators of immune cell activity and its consequential phenotypes - a more active role in inflammation regulation than previously thought. Indeed, results focusing on the immunocentric roles of peripheral lymphatic function have revealed that the basic drainage task of lymphatic vessels is a complex balance of locally processed and transported antigens as well as interstitial cytokine and immune cell signaling: an interplay that likely defines the function of IAL. This review will summarize the latest findings on how IAL impacts a series of disease states in various tissues in both preclinical models and clinical studies. This discussion will serve to highlight some emerging areas of lymphatic research in an attempt to answer the question relevant to an array of scientists and clinicians of whether IAL helps to fuel or extinguish inflammation. Impact statement Inflammatory progression is present in acute and chronic tissue pathologies throughout the body. Lymphatic vessels play physiological roles relevant to all medical fields as important regulators of fluid balance, immune cell trafficking, and immune identity. Lymphangiogenesis is often concurrent with inflammation and can potentially aide or worsen disease progression. How new lymphatic vessels impact inflammation and by which mechanism is an important consideration in current and future clinical therapies targeting inflammation and/or vasculogenesis. This review identifies, across a range of tissue-specific pathologies, the current understanding of inflammation-associated lymphangiogenesis in the progression or resolution of inflammation.

Vascular endothelial growth factor-D modulates oxidant-antioxidant balance of human vascular endothelial cells.

Vascular endothelial growth factor-D (VEGF-D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF-D stimulates the expression of proteins involved in cell-matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up-regulated expression of proteins that form the antioxidant barrier of the cell in VEGF-D-treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF-D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF-D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF-D-induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser-2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.

[Lymphangioleiomyomatosis - new concepts on pathogenesis, diagnosis and treatment]. / Limfangioleiomiomatoza- nowe poglady na patogeneze, rozpoznawanie i leczenie.

Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease presenting with cough, dyspnea on exertion and recurrent pneumothorax. Substantial achievements have been made during the past two decades regarding the pathogenesis, diagnosis and management of this disorder. LAM, affecting almost exclusively women, is associated with inactivating tuberous sclerosis complex (TSC) gene mutations in LAM cells, resulting in activation of mTOR that controls cell proliferation, growth and motility. Many parallels have been identified between LAM pathogenesis and neoplasia; inactivating mutations, the ability of LAM cells to metastasise, the induction of angiogenesis and lymphangiogenesis, invasion of the lung. Recent reports suggest that VEGF-D levels have diagnostic utility in LAM patients. Partial response of TSC-associated tumors and decrease the rate of lung function decline in females with LAM due to inhibition of mTOR pathway with sirolimus have been demonstrated.

Regulation of expression level of fms-like tyrosine kinase-4 is related to osteoclast differentiation.

INTRODUCTION: The aim of this study is to determine whether regulation of the expression level of fms-like tyrosine kinase-4 (Flt-4) is related to osteoclast differentiation. MATERIAL AND METHODS: Osteoclast formation and differentiation of mouse bone marrow cells and RAW264.7 cells were performed. To induce osteoclast differentiation, RANKL (50 ng/ml) with or without vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor-D (VEGF-D) was added to mouse bone marrow cells and RAW264.7 cells. Then cells were examined under a microscope. TRAP-positive cells with 3 nuclei or more were considered as osteoclasts and counted. The Flt-4 gene was knocked down by transfection of siRNAs against Flt-4. Immunoblot analyses were performed. RESULTS: The osteoclast formation assay indicated that VEGF-C resulted in 500 or 450 vs. 100 (p < 0.05) of osteoclasts in mouse bone marrow cells and RAW264.7 cells, respectively. Vascular endothelial growth factor-D resulted in about 600 or 630 vs. 100 (p < 0.05) of osteoclasts for both mouse bone marrow cells and RAW264.7 cells. The knock-down of Flt-4 expression abolished the induction by VEGF-C or VEGF-D, resulting in induction similar to that of the negative control PBS. CONCLUSIONS: Both VEGF-C and VEGF-D can induce osteoclast differentiation in the presence of the receptor activator of nuclear factor κB ligand. Down-regulation of expression level of Flt-4 protein abolishes osteoclast differentiation induced by VEGF-C or VEGF-D.

Lymphangioleiomyomatosis: differential diagnosis and optimal management.

Lymphangioleiomyomatosis (LAM) is an uncommon disease presented as diffuse thin-walled cystic changes in the lung. The main differential diagnoses include pulmonary Langerhans' histiocytosis (PLCH), Birt-Hogg-Dubé syndrome (BHD), lymphoid interstitial pneumonia (LIP), and amyloidosis. A combination of clinical, radiological, and pathological approaches as well as genetic testing will clarify the diagnosis in most cases. LAM is a disease almost exclusively in women. Dyspnea, pneumothorax, and hemoptysis are common presentations in LAM patients. LAM is also a lymphatic disorder affecting lymphatic vessels and lymph nodes. Chylothorax, chylous ascites, and lymphangiomyomas are frequently seen. LAM can present sporadically as a single entity or as part of tuberous sclerosis complex (TSC). Angiomyolipoma (AML) is a characteristic extra-pulmonary lesion, either found in association with sporadic or TSC-related LAM. High-risk populations should be screened for LAM, including adult women with TSC and female patients with spontaneous pneumothorax, AMLs in the kidney, and diffuse cystic lung diseases. Definitive diagnosis of LAM is based on a high level of clinical suspicion on presentation supported by pathological findings or by a distinct feature, such as a history of TSC, AMLs in the kidney, chylothorax, or chylous ascites. Vascular endothelial growth factor-D (VEGF-D) in serum is a noninvasive and reliable diagnostic biomarker. In experienced centers, trans-bronchial lung biopsy (TBLB) provides a convenient and safe way to obtain lung specimens for diagnostic purposes. An effective treatment for LAM is now available, namely using a mechanistic target of rapamycin (mTOR) inhibitor such as sirolimus. Efficacy of sirolimus has been confirmed in clinical trials. Research in other molecular-targeted therapies is under investigation. A previously little-known rare disease with no cure is now better understood with regards to its pathogenesis, diagnosis, and management. In this review, current knowledge in diagnosis and differential diagnosis of LAM will be discussed, followed by the discussion of therapy with mTOR inhibitors.