Prophylactic mesh reinforcement for midline incisional hernia prevention: systematic review and updated meta-analysis of randomized controlled trials.

BACKGROUND: Incisional hernia (IH) is a common complication after abdominal surgery. Prevention of IH is matter of intense research. Prophylactic mesh reinforcement (PMR) has been shown to be promising in the minimization of IH risk after elective midline laparotomy. METHODS: Systematic review and meta-analysis of randomized controlled trials (RCTs) comparing PMR vs. primary suture closure (PSC). Risk ratio (RR) and standardized mean difference (MD) were used as pooled effect size measures whereas 95% confidence intervals (95%CI) were used to assess relative inference. RESULTS: Fourteen RCTs (2332 patients) were included. Overall, 1280 (54.9%) underwent PMR while 1052 (45.1%) PSC. Postoperative follow-up ranged from 12 to 67 months. The incidence of IH was reduced for PMR vs. PSC (13.4% vs. 27.5%). The estimated pooled IH RR for PMR vs. PSC is 0.38 (95% CI 0.24-0.58; p < 0.001). Stratified subgroup analysis according to mesh location shows a risk reduction for intraperitoneal (RR = 0.65; 95% CI 0.48-0.89), preperitoneal (RR = 0.18; 95% CI 0.04-0.81), retromuscular (RR = 0.47; 95% CI 0.24-0.92) and onlay (RR = 0.24; 95% CI 0.12-0.51) compared to PSC. The seroma RR was higher for PMR (RR = 2.05; p = 0.0008). No differences were found for hematoma (RR = 1.49; p = 0.34), surgical site infection (SSI) (RR = 1.17; p = 0.38), operative time (OT) (MD = 0.27; p = 0.413), and hospital length of stay (HLOS) (MD = -0.03; p = 0.237). CONCLUSIONS: PMR seems effective in reducing the risk of IH after elective midline laparotomy compared to PSC in the medium-term follow-up. While the risk of postoperative seroma appears higher for PMR, hematoma, SSI, HLOS and OT seems comparable.

Paraesophageal hernia repair with laparoscopic Toupet fundoplication: impact on pulmonary function, respiratory symptoms and quality of life.

BACKGROUND: Paraesophageal hiatal hernia (PEH) is characterized by protrusion of intra-abdominal organs into the posterior mediastinum. Respiratory symptoms and reduced pulmonary function have been described as possibly related to lung compression. OBJECTIVE: To assess the effect of laparoscopic Toupet fundoplication (LTF) for PEH repair on pulmonary function, measured with pulmonary function tests (PFTs), and respiratory symptoms. METHODS: Retrospective, single-center, cohort study (November 2015-2020). All patients that completed pre- and postoperative (12 months) PFTs assessment were included. The gastroesophageal reflux disease health-related quality of life (GERD-HRQL), reflux symptom index (RSI) and short form-36 (SF-36) were used. RESULTS: Overall, 71 patients were included. The median age was 67.1 years and the majority were females (78.8%). Baseline PFTs were within normal limits in 91% of patients. At 12 month follow-up, total lung capacity (TLC) (4.77 vs. 5.07 L; p = 0.0251), vital capacity (VC) (2.97 vs. 3.31 L; p = 0.0065), forced expiratory volume in one second (FEV1) (2.07 vs. 2.44 L; p < 0.001) and forced vital capacity (FVC) (2.78 vs. 3.19 L; p < 0.001) were significantly improved. No significant differences were found for diffusing capacity of lung for carbon monoxide (DLCO) (17.09 vs. 17.24; p = 0.734), and FEV1/FVC (0.77 vs. 0.77; p = 0.967). Interestingly, improvements were more pronounced in patients with large PEH (type IIIb and IV). At 12 month follow-up, both gastrointestinal and respiratory symptoms were significantly improved and 94% of patients were satisfied with the operation. The GERD-HRQL (18.1 ± 7.9 vs. 4.01 ± 2.4; p = 0.001), RSI (37.8 ± 9.7 vs. 10.6 ± 8.9; p < 0.001) and all SF-36 items were improved. CONCLUSIONS: LTF for the treatment of PEH is safe and seems to be effective up to 12 month follow-up with improved lung volumes, spirometry values, quality of life, gastrointestinal and respiratory symptoms.

Medium-term safety and efficacy profile of paraesophageal hernia repair with Phasix-ST® mesh: a single-institution experience.

BACKGROUND: Hernia recurrence after laparoscopic repair is a perplexing problem. In an effort to reduce anatomical and clinical recurrences, different type of meshes have been used to bolster the esophageal hiatus. OBJECTIVE: The aim of this study was to assess safety, medium-term efficacy, and quality of life improvement after laparoscopic repair of hiatal hernia reinforced with a biosynthetic absorbable mesh (Phasix-ST®). METHODS: Observational single-center retrospective single-arm cohort study (November 2015-February 2021). We included all adult patients (> 18 years old) who underwent laparoscopic paraesophageal hernia repair with Phasix-ST® mesh and Toupet fundoplication. RESULTS: Sixty-eight patients were included. The median postoperative stay was 3.2 days (range 2-9) and the postoperative complication rate was 11.7%. The median follow-up time was 27 months (range 1-53). No mesh-related complications were detected. Hernia recurrence was diagnosed in six patients (8.8%). The recurrence-free probability at 34 months was 0.89 (95% CI 0.807-0.988) while at 60 months was 0.86 (95% CI 0.76-0.97). Hernia recurrences were mostly observed between 21 and 36 months after the operation. None of the patients required surgical revision and all were managed with PPI. Postoperative dysphagia requiring endoscopic balloon dilatation occurred in 2.9% of patients. Compared to baseline, both the GERD-HRQL (15.2 ± 6.2 vs. 3.2 ± 3.1; p = 0.026) and all SF-36 items were significantly improved (p < 0.001). CONCLUSIONS: Laparoscopic crura augmentation with Phasix-ST® mesh combined with a Toupet fundoplication is safe and seems effective in the medium-term follow-up. Phasix-ST® crural reinforcement resulted in low hernia recurrence rate with a sustained symptoms and quality of life improvement.

The sphingosine kinase activator K6PC-5 stimulates C2C12 myoblast differentiation.

Previously, K6PC-5, a synthetic derivative of ceramide, was demonstrated to activate sphingosine kinase (SK)-1 in keratinocytes. In this study its potential biological effect in mouse myoblasts was examined. The obtained results show that K6PC-5 promotes myogenic differentiation by enhancing myogenic marker expression, differentiation index and fusion index. Interestingly, its biological action was prevented by pharmacological inhibition of SK or S1P2 receptor, in full agreement with their recognized role in myoblast differentiation. This is the first evidence that pharmacological activation of SK accelerates myogenesis and suggests that this new therapeutic strategy could be possibly employed in skeletal muscle disorders where muscle regeneration is deficient.

An extraperitoneal approach for complex flank, iliac, and lumbar hernia.

PURPOSE: The aim of this paper is to propose our four-step technique, an open extraperitoneal approach for complex flank, lumbar, and iliac hernias. METHODS: A big polypropylene mesh is placed, covering and reinforcing all the lateral abdominal wall in an extraperitoneal space. Its borders are retroxiphoid fatty triangle and the costal arch cranially and the retropubic space caudally, psoas muscle, and paravertebral region posteriorly and contralateral rectus muscle medially. Mesh dimensions do not depend from the defect size, but prosthesis has to cover all the lateral abdominal wall. RESULTS: No major complications have been reported. The mean length of stay is 4.8 days (range 3-11). Mean follow-up is 44.8 months (range 5-92). One recurrence (4.5%) has been reported at the 1-year clinical evaluation. CONCLUSION: In conclusion, we believe that regardless size and location of the defect, every complex lateral hernia requires the same extensive repair because of the critical anatomy of the region with a big medium-heavyweight polypropylene mesh placed in an extraperitoneal plane, the only one that allows adequate covering of the visceral sac. Our technique is a safe, feasible, and reproducible treatment for this challenging surgical problem.

Prevalence and risk factors for diastasis recti abdominis: a review and proposal of a new anatomical variation.

PURPOSE: Diastasis recti abdominis (DRA) or rectus diastasis is an acquired condition in which the rectus muscles are separated by an abnormal distance along their length, but with no fascia defect. To data there is no consensus about risk factors for DRA. The aim of this article is to critically review the literature about prevalence and risk factor of DRA. METHOD: A total of 13 papers were identified. RESULTS: The real prevalence of DRA is unknown because the prevalence rate varies with measurement method, measurement site and judgment criteria, but it is certainly an extremely frequent condition. Numbers of parity, BMI, diabetes are the most plausible risk factors. We identified a new anatomical variation in cadaveric dissection and in abdominal CT image evaluation: along the semilunar line the internal oblique aponeurosis could join the rectus sheath with only a posterior layer, so without a double layer (anterior and posterior) as usually described. We conducted a retrospective review of abdominal CT images and the presence of the posterior insertion only could be considered as a risk factor for DRA. CONCLUSION: Further studies with large sample size, including nulliparous, primiparous, pluriparous and men too, are necessary for identify the real prevalence.

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as alpha-smooth muscle actin (alpha SMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated alpha SMA. More importantly, S1P challenge was responsible for the functional appearance of Ca(2+) currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P(2) turned out to be critical for the pro-differentiating effect of S1P, while S1P(3) appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration.

Original concepts in anatomy, abdominal-wall surgery, and component separation technique and strategy.

BACKGROUND: The abdominal wall can be considered comprised of two compartments: an anterior and a posterior compartment. The anterior compartment includes the anterior rectus sheath and the rectus muscle. The posterior compartment comprises the posterior rectus sheath, the transversalis fascia, and the peritoneum. When a large defect in the anterior compartment has to be corrected, for example, a rectus diastasis or large incisional hernia, an action on the anterior compartment is necessary; therefore, an anterior component separation has to be considered. If a loss of substance is present in the posterior compartment, a trasversus abdominis release should be accomplished. METHODS: We propose an original anterior compartment mobilisation, by a posterior approach. Dissection of the posterior rectus sheet proceeds until the linea semilunaris is reached. Incision of the anterior rectus sheath permits a mobilisation of the anterior compartment by a posterior approach. A mesh is placed in a sublay position. If the abdominal wall presents a loss of substance of the posterior compartment, a transversus abdominis release (TAR) can be performed in the same time. RESULTS: No hernia recurrences, no wound infection, and no mesh infection have been reported. CONCLUSIONS: The anterior compartment mobilization permits mobilization towards the midline of rectus muscle and restoration of anterior compartment, with low morbidity rate; it can be easily associated to a large sublay mesh placement, it allows the preservation of the neurovascular bundles and rectus muscle trophism, and it can be associated with a concomitant TAR procedure for the restoration of the PC, if necessary.

Open mesh vs. suture umbilical hernia repair: systematic review and updated trial sequential meta-analysis of randomized controlled trials.

PURPOSE: To examine the updated evidence on safety, effectiveness, and outcomes of mesh versus suture elective umbilical hernia (UH) repair and to explore the timely tendency variations favouring one treatment over another. METHODS: MEDLINE and CENTRAL databases were consulted. A systematic review, pairwise meta-analysis, and trial sequential analysis (TSA) were conducted. RESULTS: Six RCTs were included for a total of 742 patients. Overall, 383 (51.6%) underwent mesh, while 359 (48.4%) underwent suture repair. The estimated pooled postoperative recurrence RR was 0.27 (95% CI 0.13-0.53; p < 0.001). The TSA showed a statistically significant timely tendency in favour of mesh repair with a boundary cross curve (Z = 1.96) before reaching the information size. The estimated pooled seroma, haematoma, and wound infection RR were 1.45 (p = 0.368), 0.54 (p = 0.196), and 0.71 (p = 0.375), respectively. The TSA for wound-related complications showed partial, non-significant results. CONCLUSIONS: Elective UH mesh repair seems to be associated with reduced risk of postoperative recurrence compared to simple suture repair with a statistically significant timely trend endorsed by the TSA. Definitive considerations concerning the cumulative effect for seroma, haematoma, and wound infection are premature. Further studies are warranted to endorse these results and deeply investigate the timely tendency variations.

Pleiotropic effects of sphingolipids in skeletal muscle.

Studies of the last two decades have demonstrated that sphingolipids are important signalling molecules exerting key roles in the control of fundamental biological processes including proliferation, differentiation, motility and survival. Here we review the role of bioactive sphingolipids such as ceramide, sphingosine, sphingosine 1-phosphate, ganglioside GM3, in the regulation of skeletal muscle biology. The emerging picture is in favour of a complex role of these molecules, which appear implicated in the activation of muscle resident stem cells, their proliferation and differentiation, finalized at skeletal muscle regeneration. Moreover, they are involved in the regulation of contractile properties, tissue responsiveness to insulin and muscle fiber trophism. Hopefully, this article will provide a framework for future investigation into the field, aimed at establishing whether altered sphingolipid metabolism is implicated in the onset of skeletal muscle diseases and identifying new pharmacological targets for the therapy of multiple illnesses, including muscular dystrophies and diabetes.

Robotic inguinal hernia repair: is technology taking over? Systematic review and meta-analysis.

PURPOSE: To examine the current evidence on the therapeutic role and outcomes of robotic Transabdominal Preperitoneal Inguinal hernia repair (rTAPP) to better define its risk-benefit ratio and guide clinical decision-making. METHODS: PubMed, EMBASE, and Web of Science were consulted. A Frequentist single-arm study-level random effect meta-analysis was performed. RESULTS: Twelve studies published between 2015 and 2018 met the inclusion criteria (1645 patients). Patients' age ranged from 16 to 96, the BMI ranged from 19 to 35.6 kg/m2, and 86.1% were males. Unilateral hernia repair was performed in 69.6% while bilateral hernia repair was performed in 30.4% of patients. The operations were all conducted using the da Vinci Xi or Si robotic system (Intuitive Surgical, Inc., Sunnyvale, CA, USA). The rTAPP was successfully completed in 99.4% of patients and the operative time ranged from 45 to 180.4 min. The postoperative follow-up ranged from 16 to 368 days. The estimated pooled prevalence of intraoperative complications and conversion were 0.03% (95% CI 0.00-0.3) and 0.14% (95% CI 0.0-0.5%), respectively. The estimated pooled prevalence of urinary retention, seroma/hematoma, and overall complications were 3.5% (95% CI 1.6-5.8%), 4.1% (95% CI 1.6-7.5%), and 7.4% (95% CI 3.4-10.9%). The estimated pooled prevalence of hernia recurrence was 0.18% (95% CI 0.00-0.84%). CONCLUSIONS: Robotic technology has been progressively entering surgical thinking and gradually changing surgical procedures. Based on the results of the present study, the rTAPP seems feasible, safe, and effective in the short term for patients with unilateral and bilateral inguinal hernias. Further prospective studies and randomized controlled trials are needed to validate these findings.

Primary inguinal hernia: systematic review and Bayesian network meta-analysis comparing open, laparoscopic transabdominal preperitoneal, totally extraperitoneal, and robotic preperitoneal repair.

PURPOSE: The Open Lichtenstein technique, the Laparoscopic Trans-Abdominal PrePeritoneal (TAPP), the Totally Extra Peritoneal (TEP), and the robotic TAPP (rTAPP) are commonly performed. The aim of the present network meta-analysis was to globally compare short-term outcomes within these major surgical techniques for primary unilateral inguinal hernia repair. METHODS: PubMed, EMBASE, and Web of Science were consulted. A fully Bayesian network meta-analysis was performed. RESULTS: Sixteen studies (51.037 patients) were included. Overall, 35.5% underwent Open, 33.5% TAPP, 30.7% TEP, and 0.3% rTAPP. The postoperative seroma risk ratio (RR) was comparable considering TAPP vs. Open (RR 0.91; 95% CrI 0.50-1.62), TEP vs. Open (RR 0.64; 95% CrI 0.32-1.33), TEP vs. TAPP (RR 0.70; 95% CrI 0.39-1.31), and rTAPP vs. Open (RR 0.98; 95% CrI 0.37-2.51). The postoperative chronic pain RR was similar for TAPP vs. Open (RR 0.53; 95% CrI 0.27-1.20), TEP vs. Open (RR 0.86; 95% CrI 0.48-1.16), and TEP vs. TAPP (RR 1.70; 95% CrI 0.63-3.20). The recurrence RR was comparable when comparing TAPP vs. Open (RR 0.96; 95% CrI 0.57-1.51), TEP vs. Open (RR 1.0; 95% CrI 0.65-1.61), TEP vs. TAPP (RR 1.10; 95% CrI 0.63-2.10), and rTAPP vs. Open (RR 0.98; 95% CrI 0.45-2.10). No differences were found in term of postoperative hematoma, surgical site infection, urinary retention, and hospital length of stay. CONCLUSIONS: This study suggests that Open, TAPP, TEP, and rTAPP seem comparable in the short term. The surgical management of inguinal hernia is evolving and the effect of the adoption of innovative minimally invasive techniques should be further investigated in the long term. Ultimately, the choice of the most suitable treatment should be based on individual surgeon expertise and tailored on each patient.

Primary unilateral not complicated inguinal hernia with an effective, cheap, less invasive, and easy operation: the Trabucco repair.

BACKGROUND: In modern abdominal wall hernia surgery, the achievement of the most effective tailored repair for each specific defect with the less possible invasiveness, the quicker recovery, the lower costs and the fewer risk of local occurrences, recurrences and chronic pain is the most desirable and cutting-edge goal. METHODS: Since 1989 about 4219 primary unilateral not complicated inguinal hernias have been treated with specific indications with a sutureless and minimally invasive anterior open approach. The great majority of these procedures were performed under local anaesthesia in a day surgery regimen, with a systematic and careful nerve sparing, preservation of cremasteric muscle, and with a 3-5 cm skin incision. RESULTS: The minimally invasive sutureless nerve sparing open approach has shown a very low rate of seromas (0.45%), haematomas (0.24%) and infections (0.07%) while the width of skin incision challenges even laparoscopy. A significant reduction of both postoperative pain (2.7%) and chronic neuralgia (0.047%) has led to excellent outcomes in patients, also in terms of quality of life. Compared to the Lichtenstein's tension-free technique, which is at now the gold standard open treatment for primary inguinal hernia worldwide, there are no significant differences in the observed recurrence rate (well below 1%). CONCLUSION: In our experience of almost 30 years we have been able to experiment and refine more and more the sutureless technique proposed by Trabucco for the treatment of primitive inguinal hernia, peer to peer, improving the local anaesthesia and the ability to detect hidden defects during the repair (Spigelian included), reducing the width of the incisions and tractions on the tissues, introducing the concept of a gentle and bloodless "finger surgery" according to a minimally invasive, extremely anatomic, safe, inexpensive, very effective anterior open approach.

Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells.

The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.

Rat protein tyrosine phosphatase eta suppresses the neoplastic phenotype of retrovirally transformed thyroid cells through the stabilization of p27(Kip1).

The r-PTPeta gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPeta (the human homolog of r-PTPeta) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPeta gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPeta caused G(1) growth arrest and increased the cyclin-dependent kinase inhibitor p27(Kip1) protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPeta tumor suppressor activity is mediated by p27(Kip1) protein stabilization, because suppression of p27(Kip1) protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPeta. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27(Kip1) protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPeta regulated p27(Kip1) stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation.

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells mediated by p27kip1.

The dual-specificity phosphatase PTEN/MMAC1/TEP1 has recently been identified as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantly-inherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we confirm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced both at the mRNA and at the protein level - in five out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two different thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27kip1 and can be overcome by simultaneous co-transfection of an excess p27kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27kip1 protein was observed. In conclusion, our findings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27kip1.

Upregulation of the angiogenic factors PlGF, VEGF and their receptors (Flt-1, Flk-1/KDR) by TSH in cultured thyrocytes and in the thyroid gland of thiouracil-fed rats suggest a TSH-dependent paracrine mechanism for goiter hypervascularization.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. In vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PIGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells. Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

Modulation of in vivo growth of thyroid tumor-derived cell lines by sense and antisense vascular endothelial growth factor gene.

Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.

Key role of the cyclin-dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation.

Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin E/CDK2 (but not to cyclin D3/CDK4). When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21. Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2 activity is sufficient for growth arrest but not for cell differentiation and suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.

Regulation of fructose 2,6-bisphosphate metabolism in human fibroblasts.

In the present work, the mechanism involved in the regulation of fructose 2,6-bisphosphate (fructose-2,6-P2) metabolism in human fibroblasts has been studied. Various agents like serum, insulin and adrenaline known to affect glycolysis have been investigated for their ability to influence fructose 2,6-P2 metabolism in confluent human fibroblasts. Serum appears to be the most potent activator of fructose-2,6-P2 levels and capable of inducing a marked increase in 6-phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase), EC 2.7.1. 105). To a lesser extent insulin has the same effects. The increase in enzyme activity elicited by serum and insulin does not require de novo protein synthesis since the process is insensitive to cycloheximide. Incubation of fibroblasts in the presence of adrenaline is responsible for a significant rise in fructose-2,6-P2 levels without affecting 6-phosphofructo-2-kinase. Similar experiments performed on glucose-starved or cytochalasin B-treated cells show that the effects elicited by all the agents are strictly dependent on glucose availability.