SaeR as a novel target for antivirulence therapy against Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilizing a GFP-Lux dual reporter system driven by saeP1 promoter. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and haemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR's DNA-binding domain. When SaeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target's specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure-activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four times more potent than HR3744 and demonstrated identical antivirulence properties and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence properties, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections.

Cannabis-Induced Catatonia in a 15-Year-Old Male: A Case Report.

INTRODUCTION: Catatonia is a syndrome of primarily psychomotor disturbances most common in psychiatric mood disorders but that also rarely has been described in association with cannabis use. CASE PRESENTATION: A 15-year-old White male presented with left leg weakness, altered mental status, and chest pain, which then progressed to global weakness, minimal speech, and a fixed gaze. After ruling out organic causes of his symptoms, cannabis-induced catatonia was suspected, and the patient responded immediately and completely to lorazepam administration. DISCUSSION: Cannabis-induced catatonia has been described in several case reports worldwide, with a wide range and duration of symptoms reported. There is little known about the risk factors, treatment, and prognosis of cannabis-induced catatonia. CONCLUSIONS: This report emphasizes the importance of clinicians maintaining a high index of suspicion to accurately diagnose and treat cannabis-induced neuropsychiatric conditions, which is especially important as the use of high-potency cannabis products in young people increases.

Antivirulence Agent as an Adjuvant of ß-Lactam Antibiotics in Treating Staphylococcal Infections.

Staphylococcus aureus can cause a plethora of life-threatening infections. Antibiotics have been extensively used to treat S. aureus infections. However, when antibiotics are used at sub-inhibitory concentrations, especially for ß-lactam antibiotics, they may enhance staphylococcal pathogenicity and exacerbate the infection. The combination of antivirulence agents and antibiotics may be a novel approach to controlling antibiotic-induced S. aureus pathogenicity. We have illustrated that under in vitro conditions, antivirulence agent M21, when administered concurrently with ampicillin, suppressed the expression and production of virulence factors induced by ampicillin. In a mouse peritonitis model, M21 reduced bacterial load irrespective of administration of ampicillin. In a bacteremia model, combinatorial treatment consisting of ampicillin or ceftazidime and M21 increased the survival rate of mice and reduced cytokine abundance, suggesting the suppression of antibiotic-induced virulence by M21. Different from traditional antibiotic adjuvants, an antivirulence agent may not synergistically inhibit bacterial growth in vitro, but effectively benefit the host in vivo. Collectively, our findings from this study demonstrated the benefits of antivirulence-antibiotic combinatorial treatment against S. aureus infections and provide a new perspective on the development of antibiotic adjuvants.

Subinhibitory Concentrations of Antibiotics Exacerbate Staphylococcal Infection by Inducing Bacterial Virulence.

Antibiotics are widely used for the treatment of bacterial infections. However, injudicious use of antibiotics based on an empirical method may lead to the emergence of resistant strains. Despite appropriate administration of antibiotics, their concentrations may remain subinhibitory in the body, due to individual variations in tissue distribution and metabolism rates. This may promote bacterial virulence and complicate the treatment strategies. To investigate whether the administration of certain classes of antibiotics will induce bacterial virulence and worsen the infection under in vivo conditions. Different classes of antibiotics were tested in vitro for their ability to induce virulence in a methicillin-resistant S. aureus strain Mu3 and clinical isolates. Antibiotic-induced pathogenicity was assessed in vivo using mouse peritonitis and bacteremia models. In vitro, ß-lactam antibiotics and tetracyclines induced the expression of multiple surface-associated virulence factors as well as the secretion of toxins. In peritonitis and bacteremia models, mice infected with MRSA and treated with ampicillin, ceftazidime, or tetracycline showed enhanced bacterial pathogenicity. The release of induced virulence factors in vivo was confirmed in a histological examination. Subinhibitory concentrations of antibiotics belonging to ß-lactam and tetracycline aggravated infection by inducing staphylococcal virulence in vivo. Thus, when antibiotics are required, it is preferable to employ combination therapy and to initiate the appropriate treatment plan, following diagnosis. Our findings emphasize the risks associated with antibiotic-based therapy and underline the need for alternative therapeutic options. IMPORTANCE Antibiotics are widely applied to treat infectious diseases. Empirically treatment with incorrect antibiotics, or even correct antibiotics always falls into subinhibitory concentrations, due to dosing, distribution, or secretion. In this study, we have systematically evaluated in vitro virulence induction effect of antibiotics and in vivo exacerbated infection. The major highlight of this work is to prove the ß-lactam and tetracyclines antibiotics exacerbated disease is due to their induction effect on staphylococcal virulence. This phenomenon is common and suggests that if ß-lactam antibiotics remain the first line of defense during empirical therapy, we either need to increase patient reliability or the treatment approach may improve in the future when paired with anti-virulence drugs.

Successful Pregnancy Outcomes After Laparoscopic Management of Pheochromocytoma.

Background: Pheochromocytoma is a rare cause of hypertension in pregnancy, which is often overlooked; especially in late pregnancy because of more prevalent pre-eclampsia. It has been associated with significant morbidity and mortality rates in both mother and fetus, if not diagnosed and treated in time. Minimally invasive surgery has been infrequently used for surgical management of pheochromocytoma in pregnancy, with <20 reported cases in English literature. Case Presentation: A 26-year-old pregnant woman presented at 9 weeks of gestation with complaints of palpitations, sweating, and headache; with past history of first trimester spontaneous abortion caused by accelerated hypertension. She was found to have hypertension and diabetes, but no pedal edema, weight gain, or proteinuria. Ultrasonogram and MRI of abdomen revealed a left adrenal mass and 24 hours urinary catecholamines levels were increased, suggesting a pheochromocytoma. After preoperative optimization in consultations with obstetricians, endocrinologists, and anesthetists, she underwent laparoscopic left adrenalectomy during 15th week of gestation. Perioperative hospital course was uneventful for both mother and the fetus. After adrenalectomy, her diabetes was cured and hypertension was controlled with single antihypertensive. She was readmitted at 31 weeks of gestation with accelerated hypertension and underwent emergency caesarean for impending eclampsia at 32 weeks, and delivered a healthy female baby. 131I-meta-iodobenzylguanidine (MIBG) scan and 68Ga-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-NaI(3)-octreotide positron emission tomography-CT (68Ga-DOTANOC PET-CT) scan was obtained in postpartum period to rule out any extra-adrenal pheochromocytoma, both of which did not reveal any abnormality. At 1 year follow-up, she is normoglycemic and hypertension controlled on single antihypertensive. Conclusion: Pheochromocytoma in pregnancy is a rare but potentially lethal condition, and high index of suspicion is required for early diagnosis. Multidisciplinary coordination is required for effective management of this rare condition. Laparoscopic adrenalectomy is safe in second trimester of pregnancy for both mother and fetus.

Open surgery for pheochromocytoma: Current indications and outcomes from a retrospective cohort.

INTRODUCTION: Minimally invasive approaches are the current standard of care for pheochromocytoma/paraganglioma (PC/PG) surgery. However, a number of patients still undergo open surgery for these tumors. We evaluated the current indications and outcomes of open surgery for PC/PG to define the role of this approach. METHODS: Data of patients undergoing PC/PG surgery between July 2008 and July 2017 were retrieved from our prospectively maintained electronic database and hospital records. Tumor characteristics, operative and recovery parameters, and complications were evaluated for indications of open procedure and outcomes. RESULTS: During the study period, 106 patients underwent 124 procedures for PC/PG, including 18 simultaneous bilateral procedures. Surgeries included 102 adrenalectomies, 18 PG excisions, one partial adrenalectomy, and three partial cystectomies. Twenty-five (23.6%) patients (mean age 38.2 ± 16.1 years) underwent an open procedure, including four bilateral procedures. This included 16 adrenalectomies and 9 PG excisions. The indications for open surgery were unilateral large tumours (5; size 8-16, mean 11 cm), bilateral large tumours (2; size 6-10, mean 8.2 cm), retrocaval tumour extension (4), inter aortocaval PGs (8), Retro-mesenteric PG (1), concomitant procedures (3), and conversion from laparoscopy (2). Mean operative time was 217 ± 63.8 min, blood loss was 868 ± 734.2 ml, 11 patients required blood transfusion, and hospital stay was 6.44 ± 2.4 days. All these parameters were higher than for minimally invasive surgery (MIS) in this cohort. Three patients (12%) suffered a postoperative complication, and the rate of complications was not higher than MIS cohort (16%). CONCLUSIONS: Open surgery was most often indicated for large tumors or those located in the inter-aortocaval region. Most such procedures require large incisions and possible hepatic mobilization on the right side. The procedures can be safely completed with few complications.

Changes in blood pressure, blood sugar, and quality of life in patients undergoing pheochromocytoma surgery: a prospective cohort study.

INTRODUCTION: Pheochromocytoma surgery is associated with significant hemodynamic and metabolic changes that require post-operative monitoring. We prospectively evaluated the trends of blood pressure, blood sugar, body mass index (BMI), and quality of life (QoL) changes in a cohort of patients undergoing pheochromocytoma surgery to determine the minimum duration of monitoring and assess factors that could predict these changes. MATERIALS AND METHODS: Consecutive patients undergoing surgery for pheochromocytoma over a 20-month period were included in this ethics review board-approved, prospective cohort study. Blood pressure and sugar levels were serially monitored using a fixed protocol in the perioperative period and subsequently at 3 months after surgery. BMI and QoL (using World Health Organization Quality of Life [WHOQOL-BREF] questionnaire) were recorded at baseline and 3 months. Changes were compared and assessed for the predictive factors. RESULTS: Twenty-six patients undergoing 31 procedures were included in the study of whom 8 (30%) developed hypotension and 4 (15%) developed hypoglycemia after surgery. All hypotension episodes occurred within 6 hours of surgery. However, while 3 of the 4 patients who developed hypoglycemia manifest in the first 4 h after surgery, one occurred after 12 h. Occurrence of hypotension correlated with preoperative 24-h urinary vanillylmandelic acid (VMA) levels (P = 0.02) and the total daily dose of prazosin (P = 0.04). Out of 21 hypertensive patients, 7 (33%) had persistent hypertension (HTN) at 3 months and this was associated with age (P = 0.04) and diabetes mellitus (DM) at presentation (P = 0.04). Among six diabetic patients, 1 (16%) had persistent DM. There was significant increase in the BMI (P < 0.0001) and in WHOQOL-BREF scores postoperatively. CONCLUSIONS: Hypotension occurs in 30% patients and hypoglycemia in 15% after pheochromocytoma surgery. Hypotension occurs immediately but hypoglycemia may manifest upto 12h after surgery. Older, diabetic patients are more likely to have persistent HTN. Surgery results in increase in BMI and improvement in QoL.

Retrospective study of haemophagocytic syndrome hospitalisations in children in the USA.

INTRODUCTION: The haemophagocytic syndrome (HS) is a rare condition that presents with uncontrolled inflammation leading to multiorgan failure and is associated with significant morbidity and mortality. Current national estimates of children hospitalised due to HS are unknown. Characterising and understanding the burden of HS-related hospitalisations at a national level is the initial step in optimising the overall care. METHODS: We performed a retrospective analysis of the Nationwide Inpatient Sample (NIS) from 2012 to 2014. The NIS is the largest all-payer inpatient care dataset in the USA that contains more than seven million hospital stays and its large sample size is ideal for developing national estimates of rare conditions. All patients aged up to 18 years who were primarily hospitalised due to HS were selected for our study. Descriptive statistics were used. A multitude of patient-level and hospital-level variables were assessed. Outcome variables included overall in-hospital mortality, hospital charges and the length of stay. RESULTS: A total of 840 patients aged up to 18 years were hospitalised primarily due to HS in the USA. Mean age was 5.7 years. 57.4% were males. Whites comprised 45%. 6.5% died in hospital. A vast majority (78%) were admitted on an emergency/urgent basis. The most frequent payers included Medicaid (50%) and private insurance (36.9%). Almost 80% of children had at least one comorbid condition. 96.3% of patients were treated in urban teaching hospitals. Southern regions accounted for 42.6% of all hospitalisations. The median length of stay in hospital was 9.6 days and the median hospitalisation charge was US$100 426. CONCLUSION: Nearly 1 in 15 children who were hospitalised due to HS died. The resource utilisation associated with HS-related hospitalisations is considerable. The majority of hospitalised children with HS had comorbid conditions.

Successful use of extracorporeal membrane oxygenation in a child with obstructive shock due to massive bilateral pulmonary embolism.

BACKGROUND: Acute massive pulmonary embolism (PE) is a very rare condition in children. We report the successful use of veno-arterial extracorporeal membrane oxygenation (VA ECMO) as a lifesaving modality in a child with acute massive PE. CASE PRESENTATION: A nine-year-old female with spinal muscular atrophy type 1, chronic respiratory failure with tracheostomy and ventilator dependence presented with tachypnea and hypoxia. She had recent coiling of her pulmonary arterio-venous malformation. A chest computerized tomography scan showed massive bilateral PE. Urgent catheter-directed thrombolysis failed. She was placed on VA-ECMO with stabilization of hemodynamics. She underwent surgical thrombo-embolectomy followed by weaning of ECMO support. DISCUSSION: The use of VA ECMO supported the cardio-respiratory status and perfusion to facilitate surgical embolectomy.

Predominance of SCCmec types IV and V among biofilm producing device-associated Staphylococcus aureus strains isolated from tertiary care hospitals in Mysuru, India / Predominio de SCCmec tipos IV y V en las cepas de Staphylococcus aureus productoras de biopelículas aisladas de infecciones de dispositivos médicos en hospitales terciarios de Mysuru, India

INTRODUCTION: Device associated infections caused by Staphylococcus aureus in hospitalised patients is a serious healthcare problem. The present study was designed to determine the prevalence of biofilm-producing MRSA in device-associated infections. METHODS: Device-associated S. aureus strains (n=200) obtained from two tertiary care hospitals in Mysuru city, India were screened for biofilm production, antibiotic resistance, Panton-Valentine Leucocidin genes, SCCmec-types, spa-types, and intercellular adhesion (icaAD) dependent and independent genes. The efficacy of antibiotics (linezolid, vancomycin and rifampicin) on biofilms was studied using MTT assay, and the results were correlated with the occurrence of ica-dependent and independent factors. RESULTS: Multidrug resistance was observed in 155 strains (77.5%), and 124 strains (62%) were identified as biofilm producers. Methicillin resistance was identified in 145 strains (72.5%), and SCCmec typing of these isolates revealed high prevalence of type IV and type V. They also showed increased prevalence of pvl gene. icaAD was identified in 65 isolates, with 37 isolates showing both icaAD and ica-independent genes. spa types t852 and t657 were predominantly observed in MRSA isolates. Those isolates that had both ica-dependent and ica-independent genes showed more resistance to the screened antibiotics than the ica-dependent alone. CONCLUSION: This study reports a high prevalence of SCCmec type IV and V in biofilm producing S. aureus strains isolated from device-associated infections. Increased prevalence of pvl in SCCmec types IV and V strains suggests the role of community associated S. aureus in device-associated infections. The simultaneous presence of ica-dependent and independent genes increased the antibiotic resistance in established biofilms. Thus, S. aureus on medical devices is a potential risk for patients

INTRODUCCIÓN: Las infecciones asociadas a dispositivos médicos causadas por Staphylococcus aureus en pacientes hospitalizados son un problema importante. En el presente trabajo se estudia, en cepas de infecciones asociadas a dispositivos médicos, la prevalencia SARM productores de biopelículas y sus tipos SCCmec. MÉTODOS: Se usaron 200 cepas de S. aureus de infecciones de dispositivos médicos obtenidas de 2 hospitales terciarios de Mysuru, India. Se estudió la producción de biopelículas, los genes de la leucocidina de Panton-Valentine, los tipos SCCmec, los tipos de spa y los genes de adhesión intracelular (icaAD) dependientes e independientes. Se estudió la eficacia de linezolid, vancomicina y rifampicina en las biopelículas por un ensayo MTT y los resultados se correlacionaron con la presencia de genes ica dependientes e independientes. RESULTADOS: Ciento veinticuatro cepas (62%) producían biopelículas y se observó multirresistencia antibiótica en 155 (77,5%). Eran resistentes a meticilina 145 cepas (72,5%) y en su tipificación SCCmec se observó alta prevalencia de los tipos IV y V. Estas cepas tenían una prevalencia superior de gen pvl a las no resistentes a meticilina. icaAD se identificó en 65 aislados, de los que 37 mostraron simultáneamente genes ica dependientes e independientes. Los spa tipos t852 y t657 se observaron predominantemente en las cepas de SARM. Los aislados que tenían a la vez genes ica dependientes e ica independientes presentaban mayor resistencia a los antibióticos probados que los que tenían solo ica dependientes. CONCLUSIÓN: El presente estudio informa de una alta prevalencia de SARM de los SCCmec tipos IV y V en cepas de S. aureus productoras de biopelículas. La elevada prevalencia del gen pvl en las cepas de los SCCmec IV y V sugiere el papel de los S. aureus comunitarios en las infecciones asociadas a estos dispositivos. La presencia simultánea de genes ica dependientes e independientes aumenta la resistencia a antibióticos en las biopelículas establecidas. Por todo ello, las cepas de S. aureus en dispositivos médicos son un riesgo potencial para los pacientes

The oncogenic membrane protein LMP1 sequesters TRAF3 in B-cell lymphoma cells to produce functional TRAF3 deficiency.

Loss-of-function mutations in genes encoding the signaling protein tumor necrosis factor receptor-associated factor 3 (TRAF3) are commonly found in human B-cell malignancies, especially multiple myeloma and B-cell lymphoma (BCL). B-cell TRAF3 deficiency results in enhanced cell survival, elevated activation receptor signaling, and increased activity of certain transcriptional pathways regulating expression of prosurvival proteins. A recent analysis of TRAF3 protein staining of ∼300 human BCL tissue samples revealed that a higher proportion of samples expressing the oncogenic Epstein-Barr virus-encoded protein latent membrane protein 1 (LMP1) showed low/negative TRAF3 staining than predicted. LMP1, a dysregulated mimic of the CD40 receptor, binds TRAF3 more effectively than CD40. We hypothesized that LMP1 may sequester TRAF3, reducing its availability to inhibit prosurvival signaling pathways in the B cell. This hypothesis was addressed via 2 complementary approaches: (1) comparison of TRAF3-regulated activation and survival-related events with relative LMP1 expression in human BCL lines and (2) analysis of the impact upon such events in matched pairs of mouse BCL lines, both parental cells and subclones transfected with inducible LMP1, either wild-type LMP1 or a mutant LMP1 with defective TRAF3 binding. Results from both approaches showed that LMP1-expressing B cells display a phenotype highly similar to that of B cells lacking TRAF3 genes, indicating that LMP1 can render B cells functionally TRAF3 deficient without TRAF3 gene mutations, a finding of significant relevance to selecting pathway-targeted therapies for B-cell malignancies.

Predominance of SCCmec types IV and V among biofilm producing device-associated Staphylococcus aureus strains isolated from tertiary care hospitals in Mysuru, India.

INTRODUCTION: Device associated infections caused by Staphylococcus aureus in hospitalised patients is a serious healthcare problem. The present study was designed to determine the prevalence of biofilm-producing MRSA in device-associated infections. METHODS: Device-associated S. aureus strains (n=200) obtained from two tertiary care hospitals in Mysuru city, India were screened for biofilm production, antibiotic resistance, Panton-Valentine Leucocidin genes, SCCmec-types, spa-types, and intercellular adhesion (icaAD) dependent and independent genes. The efficacy of antibiotics (linezolid, vancomycin and rifampicin) on biofilms was studied using MTT assay, and the results were correlated with the occurrence of ica-dependent and independent factors. RESULTS: Multidrug resistance was observed in 155 strains (77.5%), and 124 strains (62%) were identified as biofilm producers. Methicillin resistance was identified in 145 strains (72.5%), and SCCmec typing of these isolates revealed high prevalence of type IV and type V. They also showed increased prevalence of pvl gene. icaAD was identified in 65 isolates, with 37 isolates showing both icaAD and ica-independent genes. spa types t852 and t657 were predominantly observed in MRSA isolates. Those isolates that had both ica-dependent and ica-independent genes showed more resistance to the screened antibiotics than the ica-dependent alone. CONCLUSION: This study reports a high prevalence of SCCmec type IV and V in biofilm producing S. aureus strains isolated from device-associated infections. Increased prevalence of pvl in SCCmec types IV and V strains suggests the role of community associated S. aureus in device-associated infections. The simultaneous presence of ica-dependent and independent genes increased the antibiotic resistance in established biofilms. Thus, S. aureus on medical devices is a potential risk for patients.

A prospective randomised controlled trial comparing chronic groin pain and quality of life in lightweight versus heavyweight polypropylene mesh in laparoscopic inguinal hernia repair.

BACKGROUND: The aim of our study was to compare chronic groin pain and quality of life (QOL) after laparoscopic lightweight (LW) and heavyweight (HW) mesh repair for groin hernia. MATERIALS AND METHODS: One hundred and forty adult patients with uncomplicated inguinal hernia were randomised into HW mesh group or LW mesh group. Return to activity, chronic groin pain and recurrence rates were assessed. Short form-36 v2 health survey was used for QOL analysis. RESULTS: One hundred and thirty-one completed follow-up of 3 months, 66 in HW mesh group and 65 in LW mesh group. Early post-operative convalescence was better in LW mesh group in terms of early return to walking (P = 0.01) and driving (P = 0.05). The incidence of early post-operative pain, chronic groin pain and QOL and recurrences were comparable. CONCLUSION: Outcomes following laparoscopic inguinal hernia repair using HW and LW mesh are comparable in the short-term as well as long-term.

Laparoscopic suturing skills acquisition: a comparison between laparoscopy-exposed and laparoscopy-naive surgeons.

BACKGROUND: Laparoscopic suturing is a difficult skill to master but can be acquired with extensive training outside the operating room. This study was done with the primary aim of assessing whether prior exposure to laparoscopic surgery helped trainees in acquiring laparoscopic suturing skills more quickly than trainees with no prior exposure to laparoscopic surgery. MATERIALS AND METHODS: Twenty laparoscopy-exposed and 20 laparoscopy-naïve surgeons performed 5 laparoscopic gastrojejunostomies each on a phantom porcine model. The performance was evaluated for operation time, overall anastomotic score (calculated by adding scores of anastomotic leak, size of the anastomosis, suture placement, and mucosal approximation), and the level of difficulty. The performance at the beginning of training (baseline) was compared to the performance at the end of training. RESULTS: All participants showed statistically significant improvement in operation time, overall anastomotic score, and difficulty level. Laparoscopy-exposed surgeons had a significantly better operation time than laparoscopynaïve surgeons at the beginning of training; however, the difference became insignificant by the end of training. The difference in overall anastomotic score was not significant between laparoscopy-exposed and naïve-surgeons. Laparoscopy-exposed surgeons showed significant improvements in anastomotic leak rate and size of the anastomosis, whereas laparoscopy naïve surgeons showed improvements in all the parameters, although these were not significant statistically. CONCLUSION: Training improves the laparoscopic suturing skills of laparoscopy-exposed as well as laparoscopy-naïve surgeons. Prior experience in laparoscopic surgery does not seem to influence the acquisition of laparoscopic suturing skills as laparoscopic-naïve surgeons manage to catch up with the skills of the laparoscopy-exposed surgeons.

Estrogen-mediated angiogenesis in thyroid tumor microenvironment is mediated through VEGF signaling pathways.

OBJECTIVES: To explore the induction of a proangiogenic phenotype in endothelial cells in the thyroid tumor microenvironment by estrogen-treated thyroid cancer cells and to define the role of vascular endothelial growth factor (VEGF) in this interaction. DESIGN: Cell-based in vitro systems analysis. SUBJECTS: Thyroid tumor cell lines (BCPAP [papillary thyroid cancer] and ML-1 [follicular thyroid cancer]) were cultured with estradiol with and without an estrogen receptor (ER) inhibitor (fulvestrant or ICI) and used to treat human umbilical vein endothelial cells (HUVECs). INTERVENTIONS: Immunofluorescence was used to confirm the presence of ERα and ERß in BCPAP cells. Conditioned medium was then used to evaluate the induction of HUVEC tubulogenesis and migration. Secretion of VEGF in this medium was evaluated by Western blot analysis. The expression of phosphoinositide 3-kinase (PI3K), the initiator of a proangiogenic pathway, was evaluated with Western blot analysis of HUVEC lysates. The subsequent effects of an ER inhibitor (fulvestrant/ICI) and a neutralizing VEGF antibody were also observed. RESULTS: Estrogen receptor α and ERß are expressed in thyroid cancer cells. Estrogen-stimulated ML-1 cells secreted an increased amount of VEGF likely as a result of ER signaling. In contact with this environment, HUVECs demonstrate enhanced tubulogenesis and migration. Western blot analysis documented estrogen-mediated upregulation of PI3K in HUVECs. These effects were mitigated by an ER inhibitor (fulvestrant/ICI) and a neutralizing VEGF antibody. CONCLUSIONS: Our data provide evidence that estrogen can induce a proangiogenic endothelial cell phenotype in the thyroid tumor microenvironment through ER and VEGF signaling. Our findings suggest that the effect of antiestrogenic therapy targeting tumor angiogenesis can be enhanced through VEGF inhibition.