Pro-inflammatory profile of preeclamptic placental mesenchymal stromal cells: new insights into the etiopathogenesis of preeclampsia.

The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia. METHODS: Chorionic villous PDMSCs were isolated from severe preeclamptic (n = 12) and physiological control term (n = 12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (n = 48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent. RESULTS: Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones. CONCLUSIONS: Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia

Synergy of Regenerative Periodontal Surgery and Orthodontics Improves Quality of Life of Patients with Stage IV Periodontitis: 24-Month Outcomes of a Multicenter RCT

In stage IV periodontitis patients with pathologic tooth migration (PTM), interdisciplinary treatment includes regenerative periodontal surgery (RPS) with an application of biomaterials and orthodontic therapy (OT) to restore function, esthetics and thereby quality of life (QoL). In a 24-month randomized trial we explored the synergy between regenerative medicine and biomechanical force application. The following methods were used: Forty-three patients had been randomized to a combined treatment comprising RPS and subsequent OT starting either 4 weeks (early OT) or 6 months (late OT) post-operatively. Clinical periodontal parameters and oral health-related QoL (GOHAI) were recorded up to 24 months. We obtained the following results: Mean clinical attachment gain (∆CAL ± SD) was significantly higher with early OT (5.96 ± 2.1 mm) versus late OT (4.65 ± 1.76 mm) (p = 0.034). Pocket closure (PPD ≤ 4 mm) was obtained in 91% of defects with early OT compared to 90% with late OT. GOHAI-scores decreased significantly from 26.1 ± 7.5 to 9.6 ± 4.7 (early OT) and 25.1 ± 7.1 to 12.7 ± 5.6 (late OT). Inconclusion, teeth severely compromised by intrabony defects and PTM can be treated successfully by RPS followed by early OT with the advantage of an overall reduced treatment time. As a result of the combined periodontal-orthodontic therapy, the oral health-related QoL of patients was significantly improved. Early stimulation of wound healing with orthodontic forces had a favorable impact on the outcomes of regenerative periodontal surgery

βhCG and Ki 67 expression in physiological placental villous explants treated by normal or PE PDMSCs conditioned media.

<p>A) βhCG mRNA (left panel) and free βhCG protein (right panel) levels in physiological villous explants treated with unconditioned [C], normal [N-cm] or preeclamptic [PE-cm] PDMSC conditioned media; B) Ki67 protein expression levels (left panel) in villous explants treated under the above mentioned experimental conditions. Figure B-right panel shows representative Western Blots for the characterization of villous explants cellular components: Cyt7 (trophoblast cell marker), CD45 (haematopoietic/endothelial cell marker) and CD166 (mesenchymal stromal cells marker). Statistical significance (*) has been considered as p<0.05.</p

Cell proliferation rate in Normal and PE-PDMSCs.

<p>Cell proliferation in Normal and PE-PDMSCs was assessed by MTT assay at time 0, 48 and 120 hours of culture. Results are expressed as means ± SE of six independent samples. Statistical significance has been considered as p<0.05. (*) = statistical significance among Normal PDMSCs time points; (**) = statistical significance between Normal and PE-PDMSCs at 120 hours of culture. (ns) = not statistically significant, referred to comparisons among PE PDMSCs time points.</p

Expression of pro-inflammatory cytokines, chemokines and growth factors in control and PE PDMSCs conditioned media.

<p>A) Scatter plot showing increased release in the culture media of pro-inflammatory cytokines and chemokines (80 molecules analyzed) by PE relative to control PDMSCs. Each data point represent the average of four control media from normal PDMSCs (white squares) and four PE PDMSCs media (black squares) used for the analysis. B) Representative cytokine array blots of unconditioned media (upper panel) and media conditioned by control (middle panel) and PE (lower panel) PDMSCs.</p

Clinical Features of the Study Population.

<p><b>A.G.A.:</b> Appropriate for Gestational Age; <b>IUGR:</b> Intra Uterine Growth Restriction; <b>CS:</b> Caesarean Section.</p

Senescence quantification in PDMSCs isolated from normal and preeclamptic placentae.

<p>(A) Normal (left panel) and PE (right panels) PDMSCs representative images after SAβ-gal nuclear staining. EB: Embryoid Body. White Arrows indicate blu nuclei positive for SAβ-gal staining, indicative of cellular senescence. (B) Quantitative analysis of cellular senescence in Normal and PE-PDMSCs. For quantitative analysis, SAβ-gal positive cells were considered to be senescent cells. The percentages of senescent cells after 48 h of incubation were determined in Normal and PE-PDMSCs populations. At least 200 cells were counted in each group. Results are expressed as means ± SE of six independent samples. Statistical significance (*) has been considered as p<0.05.</p

Lateralized overgrowth with vascular malformation caused by a somatic PTPN11 pathogenic variant: another piece added to the puzzle of mosaic RASopathies

Lateralized/segmental overgrowth disorders (LOs) encompass a heterogeneous group of congenital conditions with excessive body tissue growth. Documented molecular alterations in LOs mostly consist of somatic variants in genes of the PI3KCA/AKT/mTOR pathway or of chromosome band 11p15.5 imprinted region anomalies. In some cases, somatic pathogenic variants in genes of the RAS/MAPK pathway have been reported. We present the first case of a somatic pathogenic variant (T507K) in PTPN11 causing a LO phenotype characterized by severe lateralized overgrowth, vascular proliferation, and cerebral astrocytoma. The T507K variant was detected in DNA from overgrown tissue in a leg with capillary malformation. The astrocytoma tissue showed a higher PTPN11 variant allele frequency. A pathogenic variant in FGFR1 was also found in tumor tissue, representing a second hit on the RAS/MAPK pathway. These findings indicate that RAS/MAPK cascade overactivation can cause mosaic overgrowth phenotypes resembling PIK3CA-related overgrowth disorders (PROS) with cancer predisposition and are consistent with the hypothesis that RAS/MAPK hyperactivation can be involved in the pathogenesis of astrocytoma. This observation raises the issue of cancer predisposition in patients with RAS/MAPK pathway gene variants and expands genotype spectrum of LOs and the treatment options for similar cases through inhibition of the RAS/MAPK oversignalling