Proteomics reveals ablation of PlGF increases antioxidant and neuroprotective proteins in the diabetic mouse retina.

Placental growth factor (PlGF or PGF), a member of the vascular endothelial growth factor (VEGF) sub-family, plays a crucial role in pathological angiogenesis and inflammation. However, the underlying molecular mechanisms that PlGF mediates regarding the complications of non-proliferative diabetic retinopathy (DR) remain elusive. Using an LC-MS/MS-based label-free quantification proteomic approach we characterized the alterations in protein expression caused by PlGF ablation in the retinas obtained from C57BL6, Akita, PlGF-/- and Akita.PlGF-/- mice. After extraction and enzymatic digestion with Trypsin/LysC, the retinal proteins were analyzed by Q-Exactive hybrid Quadrupole-Orbitrap mass spectrometry. Differentially expressed proteins (DEPs) were identified in four comparisons based on Z-score normalization and reproducibility by Pearson's correlation coefficient. The gene ontology (GO), functional pathways, and protein-protein network interaction analysis suggested that several proteins involved in insulin resistance pathways (Gnb1, Gnb2, Gnb4, Gnai2, Gnao1, Snap2, and Gngt1) were significantly down-regulated in PlGF ablated Akita diabetic mice (Akita.PlGF-/- vs. Akita) but up-regulated in Akita vs. C57 and PlGF-/- vs. C57 conditions. Two proteins involved in the antioxidant activity and neural protection pathways, Prdx6 and Map2 respectively, were up-regulated in the Akita.PlGF-/- vs. Akita condition. Overall, we predict that down-regulation of proteins essential for insulin resistance, together with the up-regulation of antioxidant and neuroprotection proteins highlight and epitomize the potential mechanisms important for future anti-PlGF therapies in the treatment of DR.

Loss of placental growth factor ameliorates maternal hypertension and preeclampsia in mice.

Preeclampsia remains a clinical challenge due to its poorly understood pathogenesis. A prevailing notion is that increased placental production of soluble fms-like tyrosine kinase-1 (sFlt-1) causes the maternal syndrome by inhibiting proangiogenic placental growth factor (PlGF) and VEGF. However, the significance of PlGF suppression in preeclampsia is uncertain. To test whether preeclampsia results from the imbalance of angiogenic factors reflected by an abnormal sFlt-1/PlGF ratio, we studied PlGF KO (Pgf-/-) mice and noted that the mice did not develop signs or sequelae of preeclampsia despite a marked elevation in circulating sFLT-1. Notably, PlGF KO mice had morphologically distinct placentas, showing an accumulation of junctional zone glycogen. We next considered the role of placental PlGF in an established model of preeclampsia (pregnant catechol-O-methyltransferase-deficient [COMT-deficient] mice) by generating mice with deletions in both the Pgf and Comt genes. Deletion of placental PlGF in the context of COMT loss resulted in a reduction in maternal blood pressure and increased placental glycogen, indicating that loss of PlGF might be protective against the development of preeclampsia. These results identify a role for PlGF in placental development and support a complex model for the pathogenesis of preeclampsia beyond an angiogenic factor imbalance.

Effects of placental growth factor deficiency on behavior, neuroanatomy, and cerebrovasculature of mice.

Preeclampsia, a hypertensive syndrome occurring in 3-5% of human pregnancies, has lifelong health consequences for fetuses. Cognitive ability throughout life is altered, and adult stroke risk is increased. One potential etiological factor for altered brain development is low concentrations of proangiogenic placental growth factor (PGF). Impaired PGF production may promote an antiangiogenic fetal environment during neural and cerebrovascular development. We previously reported delayed vascularization of the hindbrain, altered retinal vascular organization, and less connectivity in the circle of Willis in Pgf-/- mice. We hypothesized Pgf-/- mice would have impaired cognition and altered brain neuroanatomy in addition to compromised cerebrovasculature. Cognitive behavior was assessed in adult Pgf-/- and Pgf+/+ mice by four paradigms followed by postmortem high-resolution MRI of neuroanatomy. X-ray microcomputed tomography imaging investigated the three-dimensional cerebrovascular geometry in another cohort. Pgf-/- mice exhibited poorer spatial memory, less depressive-like behavior, and superior recognition of novel objects. Significantly smaller volumes of 10 structures were detected in the Pgf-/- compared with Pgf+/+ brain. Pgf-/- brain had more total blood vessel segments in the small-diameter range. Lack of PGF altered cognitive functions, brain neuroanatomy, and cerebrovasculature in mice. Pgf-/- mice may be a preclinical model for the offspring effects of low-PGF preeclampsia gestation.

Deoxycorticosterone acetate-salt hypertension activates placental growth factor in the spleen to couple sympathetic drive and immune system activation.

AIMS: Chronic increase of mineralocorticoids obtained by administration of deoxycorticosterone acetate (DOCA) results in salt-dependent hypertension in animals. Despite the lack of a generalized sympathoexcitation, DOCA-salt hypertension has been also associated to overdrive of peripheral nervous system in organs typically targeted by blood pressure (BP), as kidneys and vasculature. Aim of this study was to explore whether DOCA-salt recruits immune system by overactivating sympathetic nervous system in lymphoid organs and whether this is relevant for hypertension. METHODS AND RESULTS: To evaluate the role of the neurosplenic sympathetic drive in DOCA-salt hypertension, we challenged splenectomized mice or mice with left coeliac ganglionectomy with DOCA-salt, observing that they were both unable to increase BP. Then, we evaluated by immunofluorescence and ELISA levels of the placental growth factor (PlGF) upon DOCA-salt challenge, which significantly increased the growth factor expression, but only in the presence of an intact neurosplenic sympathetic drive. When PlGF KO mice were subjected to DOCA-salt, they were significantly protected from the increased BP observed in WT mice under same experimental conditions. In addition, absence of PlGF hampered DOCA-salt mediated T cells co-stimulation and their consequent deployment towards kidneys where they infiltrated tissue and provoked end-organ damage. CONCLUSION: Overall, our study demonstrates that DOCA-salt requires an intact sympathetic drive to the spleen for priming of immunity and consequent BP increase. The coupling of nervous system and immune cells activation in the splenic marginal zone is established through a sympathetic-mediated PlGF release, suggesting that this pathway could be a valid therapeutic target for hypertension.

PlGF knockdown inhibited tumor survival and migration in gastric cancer cell via PI3K/Akt and p38MAPK pathways.

The molecular signalling of placental growth factor (PlGF), a member of the vascular endothelial growth factor family, was not uncovered in human adenocarcinoma gastric cell line (AGS). The purpose of this study was to examine the inhibitory effects of PlGF knockdown on cell proliferation, apoptosis and migration through p38 mitogen-activated protein kinase (p38MAPK) and PI3K pathways in human adenocarcinoma gastric cell line (AGS). To study PlGF knockdown effect, AGS cells were treated with 40 pmol of small interfering RNA (siRNA) related to PlGF gene and also a scrambled siRNA as control. Trypan Blue and Anexin V staining of AGS cells treated with PlGF-specific siRNA showed induction of apoptosis. Wound healing assay and zymography indicated that cellular migration and matrix metalloproteinases activities were reduced in response to PlGF knockdown. Phosphorylation of Akt and p38MAPK was reduced in AGS cells treated with PlGF-specific siRNA. PlGF knockdown decreased transcripts of PI3K, Akt, p38MAPK, PCNA, Caspase-3, OCT3/OCT4 and CD44, but elevated p53 and SOX2 transcripts. Our results indicated that PlGF knockdown decreased migration and induced apoptosis through PI3K/Akt1 and p38MAPK signal transduction in AGS cells.