TRPM3 as a novel target to alleviate acute oxaliplatin-induced peripheral neuropathic pain.

ABSTRACT: Chemotherapy-induced peripheral neuropathic pain (CIPNP) is an adverse effect observed in up to 80% of patients of cancer on treatment with cytostatic drugs including paclitaxel and oxaliplatin. Chemotherapy-induced peripheral neuropathic pain can be so severe that it limits dose and choice of chemotherapy and has significant negative consequences on the quality of life of survivors. Current treatment options for CIPNP are limited and unsatisfactory. TRPM3 is a calcium-permeable ion channel functionally expressed in peripheral sensory neurons involved in the detection of thermal stimuli. Here, we focus on the possible involvement of TRPM3 in acute oxaliplatin-induced mechanical allodynia and cold hypersensitivity. In vitro calcium microfluorimetry and whole-cell patch-clamp experiments showed that TRPM3 is functionally upregulated in both heterologous and homologous expression systems after acute (24 hours) oxaliplatin treatment, whereas the direct application of oxaliplatin was without effect. In vivo behavioral studies using an acute oxaliplatin model for CIPNP showed the development of cold and mechano hypersensitivity in control mice, which was lacking in TRPM3 deficient mice. In addition, the levels of protein ERK, a marker for neuronal activity, were significantly reduced in dorsal root ganglion neurons derived from TRPM3 deficient mice compared with control after oxaliplatin administration. Moreover, intraperitoneal injection of a TRPM3 antagonist, isosakuranetin, effectively reduced the oxaliplatin-induced pain behavior in response to cold and mechanical stimulation in mice with an acute form of oxaliplatin-induced peripheral neuropathy. In summary, TRPM3 represents a potential new target for the treatment of neuropathic pain in patients undergoing chemotherapy.

Mimicking Sampson's Retrograde Menstrual Theory in Rats: A New Rat Model for Ongoing Endometriosis-Associated Pain.

Endometriosis is a prevalent gynecologic disease, defined by dysfunctional endometrium-like lesions outside of the uterine cavity. These lesions are presumably established via retrograde menstruation, i.e., endometrial tissue that flows backwards during menses into the abdomen and deposits on the organs. As ongoing pain is one of the main pain symptoms of patients, an animal model that illuminates this problem is highly anticipated. In the present study, we developed and validated a rat model for ongoing endometriosis-associated pain. First, menstrual endometrial tissue was successfully generated in donor rats, as validated by gross examination, histology and qPCR. Next, endometriosis was induced in recipient animals by intraperitoneal injection of menstrual tissue. This resulted in neuro-angiogenesis as well as established endometriosis lesions, which were similar to their human counterparts, since epithelial and stromal cells were observed. Furthermore, significant differences were noted between control and endometriosis animals concerning bodyweight and posture changes, indicating the presence of ongoing pain in animals with endometriosis. In summary, a rat model for endometriosis was established that reliably mimics the human pathophysiology of endometriosis and in which signs of ongoing pain were detected, thus providing a new research tool for therapy development.

Functional Expression of TRP Ion Channels in Endometrial Stromal Cells of Endometriosis Patients.

Endometriosis is a common gynecological disease that is characterized by the presence of functional endometrial-like lesions in the abdominal cavity. Aside from epithelial cells, these lesions consist of stromal cells that have the capacity to migrate, adhere, proliferate, and induce neuro- and lymphangiogenesis, which allows them to survive at ectopic locations. However, the exact underlying mechanisms that regulate these changes are yet to be elucidated. The common ground of these processes, however, is the second messenger, calcium. In this regard, members of the superfamily of transient receptor potential (TRP) ion channels, which are known to be calcium-permeable and expressed in the endometrium, have emerged as key regulators. Here, we assessed the molecular and functional expression of TRP channels in stromal cells isolated from the eutopic endometrium of endometriosis patients and controls. Using RT-qPCR, high mRNA levels of TRPV2, TRPV4, TRPM4, TRPM7, TRPC1, TRPC3, TRPC4, and TRPC6 were observed in the whole endometrium throughout the menstrual cycle. Additionally, and in line with previous reports of control patients, TRPV2, TRPV4, TRPC1/4, and TRPC6 were present in human endometrial stromal cells (hESC) from endometriosis patients both at the molecular and functional level. Moreover, proliferation and migration assays illustrated that these parameters were not affected in stromal cells from endometriosis patients. Furthermore, comparison between eutopic and ectopic endometrial samples revealed that the RNA expression pattern of TRP channels did not differ significantly. Collectively, although a functional expression of specific ion channels in hESCs was found, their expression did not correlate with endometriosis.

Acute Drug Effects on the Human Placental Tissue: The Development of a Placental Murine Xenograft Model.

OBJECTIVE: A pilot study was conducted to establish a human placental xenograft, which could serve as a model to evaluate the effect of toxic exposures during pregnancy. STUDY DESIGN: The protocol consisted of engraftment of third-trimester human placental tissue in immunocompromised mice, after induction of a pseudo-pregnancy state by ovariectomy and progesterone supplementation. To validate the model, the placental tissue before and after engraftment was examined by immunohistochemistry, fluorescence-activated cell sorting (FACS), single-nucleotide polymorphism (SNP) genotyping, and whole transcriptome sequencing (WTSS). The human chorion gonadotropin (hCG) production in serum and urine was examined by enzyme-linked immunosorbent assay. RESULTS: Microscopic evaluation of the placental tissue before and after engraftment revealed a stable morphology and preserved histological structure of the human tissue. Viable trophoblast was present after engraftment and remained stable over time. Vascularization and hormonal secretion (hCG) were present till 3 weeks after engraftment. Thirty-one SNPs were equally present, and there was a stable expression level for 56 451 genes evaluated by whole transcriptome sequencing. CONCLUSION: Although this human placental xenograft model cannot copy the unique uterine environment in which the placenta develops and interacts between the mother and the fetus, it could be a suitable tool to evaluate the acute impact and adaptive processes of the placental tissue to environmental changes.

Increased expression of placental growth factor in high-grade endometrial carcinoma.

Placental growth factor (PlGF), a homolog of vascular endothelial growth factor (VEGF), exerts pleiotropic functions in cancer by affecting tumor cells as well as endothelial and inflammatory cells. Moreover, PlGF expression correlates with tumor stage, recurrence, metastasis and patient outcome in different types of cancer. Recently, administration of anti-PlGF therapy reduced tumor growth and metastasis in preclinical tumor models. In the present study, we evaluated the diagnostic and prognostic value of systemic and local expression of PlGF in primary endometrial carcinomas. PlGF levels in tumor lysates (n=128) and serum (n=88) of patients with primary endometrial cancer were determined using ELISA. PlGF mRNA expression in endometrial carcinoma tissues was quantified by quantitative qRT-PCR. Results were compared to endometrial cancer stage and grade. Systemic PlGF levels were not altered in patients with endometrial cancer (FIGO stage I-II-III) as compared to healthy controls. Only in FIGO stage IV patients, serum PlGF levels were slightly increased. Local PlGF mRNA and protein expression in endometrial tumors progressively increased with tumor grade. In endometrioid carcinomas, PlGF mRNA expression was significantly increased in endometrioid grade 3 tumors as compared to normal endometrial tissue. PlGF protein expression was significantly increased in endometrioid grade 2 and 3 carcinomas and in serous carcinomas as compared to normal endometrial tissue. Our study showed that systemic/serum PlGF levels cannot be used as a diagnostic or prognostic marker in endometrial cancer. However, the increased local expression of PlGF, primarily in high-grade carcinomas, underscores the possibility for preclinical assessment of anti-PlGF therapy in endometrial cancer.

Adverse adipose phenotype and hyperinsulinemia in gravid mice deficient in placental growth factor.

Pregnancy-induced metabolic changes are regulated by signals from an expanded adipose organ. Placental growth factor (PlGF), acting through vascular endothelial growth factor receptor-1, may be among those signals. There is a steep rise in circulating PlGF during normal pregnancy, which is repressed in gravidas who develop preeclampsia. PlGF-deficiency in mice impairs adipose vascularization and development. Here we studied young-adult PlGF-deficient (PlGF(-/-)) and wild-type mice on a high-fat diet in the nongravid state and at embryonic day (E) 13.5 or E18.5 of gestation. Litter size and weight were normal, but E18.5 placentas were smaller in PlGF(-/-) pregnancies. PlGF(-/-) mice showed altered intraadipose dynamics, with the following: 1) less blood vessels and fewer brown, uncoupling protein (UCP)-1-positive, adipocytes in white sc and perigonadal fat compartments and 2) white adipocyte hypertrophy. The mRNA expression of beta(3)-adrenergic receptors, peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and UCP-1 was decreased accordingly. Moreover, PlGF(-/-) mice showed hyperinsulinemia. Pregnancy-associated changes were largely comparable in PlGF(-/-) and wild-type dams. They included expanded sc fat compartments and adipocyte hypertrophy, whereas adipose expression of key angiogenesis/adipogenesis (vascular endothelial growth factor receptor-1, peroxisome proliferator-activated receptor-gamma(2)) and thermogenesis (beta(3)-adrenergic receptors, peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and UCP-1) genes was down-regulated; circulating insulin levels gradually increased during pregnancy. In conclusion, reduced adipose vascularization in PlGF(-/-) mice impairs adaptive thermogenesis in favor of energy storage, thereby promoting insulin resistance and hyperinsulinemia. Pregnancy adds to these changes by PlGF-independent mechanisms. Disturbed intraadipose dynamics is a novel mechanism to explain metabolic changes in late pregnancy in general and preeclamptic pregnancy in particular.

Diet-induced obesity in gravid rats engenders early hyperadiposity in the offspring.

Exposure to a dysmetabolic in utero environment may be one of the mechanisms to explain why individuals with high birth weight are more likely to remain overweight. We explored this hypothesis in an animal model of diet-induced obesity (DIO). We studied adipose tissue development and glucose tolerance in the offspring of rat dams fed a diet rich in milk and sugar from early adulthood until day (d) 2 postpartum. This diet promoted body weight (BW) gain and was previously shown to produce insulin resistance and gestational glucose intolerance. The DIO offspring showed a higher BW in early life (between d7 and d35), with a maximum of 1 SD above the mean BW of controls; however, BW in DIO offspring after d35 was comparable with that of controls. Neonatal DIO offspring also showed larger fat depots, adipocyte hypertrophy (P

Adipose tissue in offspring of Lepr(db/+) mice: early-life environment vs. genotype.

Gravidas with obesity and diabetes ("diabesity") may transmit this syndrome to their children through genetic and nongenetic mechanisms. Here, we used the Lepr(db/+) diabese mouse to examine the magnitude of these transmission modes, focusing on adipose tissue (AT). We compared the following six groups: wild-type (+/+) offspring from +/+ or db/+ dams (different early life environment) and db/+ offspring from db/+ dams, fed a standard or high-fat diet. Weight gain (0-8 wk) was higher in +/+ offspring from db/+ vs. +/+ mothers, and even higher in db/+ vs. +/+ offspring from db/+ mothers. In addition, we observed a stepwise increase in AT and adipocyte size in +/+ from +/+ mice, +/+ from db/+ mice, and db/+ mice at 8 wk. Differences in weight and adiposity between +/+ offspring from db/+ vs. +/+ dams were more pronounced in males than in females. Leptin and apelin mRNA levels in white and brown AT were higher in +/+ offspring from db/+ vs. +/+ dams; however, leptin, apelin, and tumor necrosis factor-alpha expression were boosted more robustly in db/+ offspring. The high-fat diet amplified AT differences between db/+ vs. +/+ offspring from db/+ dams, but not between +/+ offspring from db/+ vs. +/+ dams. Moreover, db/+ but not +/+ offspring from db/+ mothers were insulin-resistant and hyperinsulinemic after a glucose challenge. In conclusion, the genetic transmission of the diabesity phenotype clearly prevailed, but the early-life diabesity environment had discernible effects on postnatal weight gain as well as on adipocyte size and adipokine expression at a postpubertal age.

Chronic tumor necrosis factor-alpha infusion in gravid C57BL6/J mice accelerates adipose tissue development in female offspring.

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is thought to mediate, in part, the link between obesity and insulin resistance, and women with gestational diabetes mellitus (GDM) have raised serum TNF-alpha concentrations. Our objective was to investigate whether systemic TNF-alpha administration into gravid C57BL6/J mice causes a GDM-like syndrome and affects growth and adipose tissue (AT) development in the offspring. METHODS: We assessed glucose tolerance and reproductive outcome in mice infused with saline, or 2 mug or 4 mug recombinant mouse (rm)TNF-alpha by subcutaneous mini-osmotic pumps between days (d)11.5 and 18.5 of gestation. Subsequently, we studied the effects of the 2-mug dose on maternal AT metabolism. Finally, the growth of offspring exposed to 2 mug rmTNF-alpha in utero was followed until 8 weeks postnatal age. At 8 weeks, we assessed AT accumulation, as well as adipocyte area in white AT and insulin sensitivity in males, and adipokine mRNA levels in various AT depots in females. RESULTS: The peak glucose response to an intraperitoneal glucose stimulus in late-gravid mice and fetal weight were higher with 2 mug but not 4 mug rmTNF-alpha compared with saline; however, 2 mug TNF-alpha did not affect AT parameters. The female but not male offspring of these mice showed accelerated growth, hyperadiposity, robustly increased leptin expression in all AT depots, and raised fasting blood glucose. CONCLUSIONS: TNF-alpha infusion (2 mug for 7 days) in gravid mice resulted in a mild GDM syndrome and accelerated AT development in the offspring in a sex-specific manner. The data suggest that TNF-alpha mediates in part the effects of GDM on fetal growth and postnatal adiposity, and constitutes a potential mediator of intrauterine programming.

Maternal body size and birth weight: can insulin or adipokines do better?

Overweight gravidas and gravidas with a robust weight gain have an accrued risk of delivering a large-for-gestational age (LGA) baby. Here, we examined whether the measurement of insulin and adipokines--peptides secreted mainly by adipose tissue--at the glucose challenge test (GCT) improves the prediction of birth weight. We studied 631 singleton pregnancies at 24 to 29 weeks' gestational age (GA) with data on height, baseline body weight (BW), and BW change between baseline and the GCT. In addition to glucose and insulin, we measured adiponectin, leptin, soluble leptin receptor (the main leptin-binding protein), and tumor necrosis factor alpha. We found that birth weight was related to maternal height, baseline BW, and BW change, and also--albeit less strongly--to insulin, adiponectin, leptin, and soluble leptin receptor concentrations. In multiple regression analyses, body size parameters explained approximately 10% of the variance in birth weight, of which BW change was the most important correlate, but the metabolic markers added only approximately 2% variance, with leptin alone adding 1.4%. Gravidas carrying a small-for-GA (SGA) fetus were more likely to have a leptin value in the highest quartile than those with an appropriate-for-GA fetus (odds ratio, 2.6; 95% confidence interval, 1.1-6.3; P = .04), but there were no other differences in the metabolic markers between SGA or LGA and appropriate-for-GA pregnancies. In conclusion, measuring insulin and adipokines at the GCT has limited, if any, clinical benefit to predict which fetuses will be SGA or LGA at birth.

Adipokine profile and C-reactive protein in pregnancy: effects of glucose challenge response versus body mass index.

OBJECTIVE: To test the hypothesis that gravidas who have an abnormal response to glucose loading have dysfunctional adipose tissue cells that produce more insulin resistance-inducing and proinflammatory adipokines but less insulin-sensitizing adipokines. METHODS: We performed a nested case-control study within a larger sample of gravidas who had a glucose challenge test (GCT) at 24-29 weeks; we compared 73 cases with an abnormal GCT (>8.3 mM) and 146 controls with a strictly normal GCT (<7.2 mM) matched for body mass index (BMI) and height (mean difference between cases and controls: 0.1 kg/m(2) and 1 cm, respectively). We measured plasma insulin, adipokines (leptin, adiponectin, resistin, tumor necrosis factor [TNF]-alpha, interleukin [IL]-6), soluble leptin receptor (sOb-R), the main leptin-binding protein, and C-reactive protein (CRP). RESULTS: The cases showed a 48% increase in insulin concentrations and a 27% increase in TNF-alpha concentrations compared to the controls (both P < .0001), but leptin, sOb-R, IL-6, and adiponectin, as well as CRP, concentrations were comparable between cases and controls. In the whole group (n = 219), BMI was correlated with insulin, leptin, IL-6, and CRP, and inversely with sOb-R and adiponectin concentrations (all P < .0003). CONCLUSIONS: Plasma leptin, sOb-R, IL-6, and adiponectin, as well as CRP, are strongly related to BMI in gravidas at 24-29 weeks gestational age but not to the glucose loading response. However, TNF-alpha is higher in women with an abnormal GCT. Further studies should disclose the source of increased TNF-alpha in these women, and to assess whether TNF-alpha is causally related to glucose intolerance during pregnancy.

Pregnancy in mice lacking the vitamin D receptor: normal maternal skeletal response, but fetal hypomineralization rescued by maternal calcium supplementation.

Fetal mineralization appears to be driven by the pregnancy-induced stimulation of intestinal Ca absorption. We thus hypothesized that mineralization would be impaired in fetuses of mice that lack the vitamin D receptor (VDR). Here we report on the maternal response to pregnancy, and the fetal mineralization, in mice with a homozygous disruption of the VDR gene (VDR-/-) mated with wild-type (wt) males. We found that VDR-/- mice show mild hypocalcemia, clear rickets and osteomalacia on bone histomorphometry, lower cortical bone density on quantitative tomography, and reduced concentrations of calbindin-D9k (CaBP-D9k) in duodenal mucosa and kidney. The skeletal response to pregnancy was comparable in wt and VDR-/- mice; duodenal CaBP-D9k concentrations increased during pregnancy in VDR-/- as in wt mice, but remained 40% lower than in wt mice. We confirmed our hypothesis that mineralization is defective in d18.5 VDR+/- fetuses of VDR-/- mice, both by whole-body Ca determination and histomorphometric evaluation; the number of osteoclastic cells in bone was increased. The fetuses were hypercalcemic and had a 5-fold increase in circulating 1,25(OH)2D3. We then studied pregnancies in VDR-/- females, mated with wt males, fed a high Ca/P/lactose rescue diet during pregnancy. The rescue diet normalized the mineralization, the number of osteoclastic cells, and plasma Ca and 1,25(OH)2D3 concentrations in the fetuses. We interpret the data as evidence that, to ensure normal fetal mineralization, the maternal VDR-dependent intestinal Ca absorption can be substituted by passive Ca absorption entrained by a higher Ca intake. Alternatively or additionally, elevated 1,25(OH)2D3 in utero may disturb bone development.