The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes

In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap 19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap 19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations

Targeting MAPK phosphorylation of Connexin43 provides neuroprotection in stroke

Connexin43 (Cx43) function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of wild-type (WT) and knock-in Cx43 with serine to alanine mutations at the protein kinase C (PKC) site Cx43(S368A), the casein kinase 1 (CK1) sites Cx43(S325A/328Y/330A), and the mitogen-activated protein kinase (MAPK) sites Cx43(S255/262/279/282A) (MK4) on a permanent middle cerebral artery occlusion (pMCAO) stroke model. We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume that was associated with improvement in behavioral performance. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. Pharmacological blockade of Cx43 hemichannels with TAT-Gap19 also significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function

Epidermoid Polycystic Ovaries and Uterine Hypoplasia in a Cow

 Background: Ovarian cysts originate from mature follicles that do not ovulate within the predicted time according to the oestrous cycle. Dermoid cysts are structures lined by keratinized stratified squamous epithelium, without adjacent epidermal structures, filled by keratinic debris and amorphous proteinaceus material. They are small, located below the cortex, near the hilus. There is controversy regarding the teratogenicity of the dermoid cyst. Some authors use the nomenclature of benign cystic ovarian teratoma, others, reports that although the fibrous wall and histological features are similar to teratomas, it is not associated with this germ cell-derived neoplasia.Case: Ovaries and uterus were received from a female, adult bovine, Nelore, from a slaughterhouse. The left ovary measured 6.5x5.0x3.9 cm and weighed 80 g; the right ovary measured 5.7x3.7x3.0 cm and weighed 60 g. Grossly, the ovaries were similar, presenting floating consistency, multilobulated and pointed aspect, and at the cut surface, there was extravasation of mucopurulent content of whitish colour. Regarding to the uterus, macroscopically, the uterine horns were infantile and decrease in the diameter. Fragments of the tissues were collected and fixed in 10% buffered formalin for histopathological examination and the staining of the slides was done with hematoxylin and eosin. Microscopically, in the ovaries, it was observed rare vestigial elements of ovary identifying an atresic follicle. Polycystic formation with cysts exhibiting partial or total coating of a keratinized squamous epithelium was also observed. In the lumen abundant keratinous material was present. The uterine microscopy revealed compact endometrium with areas variably infiltrated by lymphocytes, plasmocytes and occasional neutrophils. Areas devoid of endometrial glands were observed, in addition to other areas with reduced quantity and size. Also, the myoepithelial layer was thin and enlargement of the endometrial vascular space was noted.Discussion: Only one study was found reporting three cases of unilateral dermoid cyst in bovine ovary, also from slaughterhouse. Dermoid cysts appear most often in Zebu cattle. Clinically, these cysts do not necessarily result in infertility and do not interfere in the ovarian functions, due to this, the ovaries are not referred for histopathology examination, which difficult its classification. However, in this study the histological examination showed the absence of follicular development, which characterizes infertility, including endocrine dysfunction. Macroscopically ovarian dermoid cysts are similar to abscesses. It has a viscous or milky content inside and, at the cut surface, usually the content project to the surface. Histologically, there are dilated cysts containing material in the lumen at various stages of keratinization conferring a lamellar aspect and a keratinized stratified squamous epithelium. In this study, we observed pseudostratified epithelial lining containing cilia in addition to lumen keratin content, without follicles or adnexal structures, corroborating the findings in the literature. Also, it was observed uterine hypoplasia, which occurs due to failure of the development of the paramesonephric ducts during embryonic development. Ovarian dermoid cysts associated with uterine hypoplasia are infrequent in animals and, clinically, may not present changes in fertility and ovarian function

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Mechanisms of Hypoxia-Induced Neurovascular Remodeling in PlGF Knockout Mice

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain

Role of PlGF in hypoxia-induced brain

Vascular endothelial growth factor (VEGF) mediator family and their receptors are involved in co-ordinated regulation of angiogenesis and neurogenesis after stroke 1. We have recently shown that placenta-derived growth factor (PlGF), a ligand for both VEGFR1 and neuropilin-1 (NP-1), plays a permissive role for VEGF- and astrocyte-induced angiogenic responses of human brain endothelial cells (BEC) 2. The role of PlGF in hypoxia-induced angiogenesis remains unclear.Peer reviewed: YesNRC publication: Ye

VEGFR-2-mediated increased proliferation and survival in response to oxygen and glucose deprivation in PlGF knockout astrocytes: J.Neurochem.

In hypoxic/ischemic conditions, astrocytes are involved in neuroprotection and angiogenesis. Vascular endothelial growth factor (VEGF) induces angiogenesis and exhibits neuroprotective and neurotrophic properties. However, the role of placental growth factor (PlGF), a VEGF homolog, in these processes is unclear. Therefore, proliferation and survival studies were performed on PlGF knockout (PlGF-/-) and wild-type (PlGF+/+) mouse astrocytes. A significant increase in cell proliferation and survival to oxygen and glucose deprivation (OGD) was observed in PlGF-/- compared to PlGF+/+ astrocytes. Interestingly, no PlGF protein expression was detected in PlGF+/+ astrocytes and no changes in VEGF protein levels were observed between the two genotypes. Real-time PCR and immunocytochemistry showed over-expression of VEGF receptor-2 (VEGFR-2) in PlGF-/- compared with PlGF+/+ astrocytes. Confocal microscopy revealed nuclear, membrane, and cytoplasmic localization of VEGFR-2. In vivo over-expression of VEGFR-2 mRNA was also detected in PlGF-/- compared with PlGF+/+ astrocytes. Stimulation with VEGF165 resulted in increased proliferation in PlGF-/- compared with PlGF+/+ astrocytes. This effect was blocked by the VEGFR-2 antagonist, VEGF165b. The enhanced proliferation of PlGF-/- astrocytes correlated with increased phospho-extracellular-signal-regulated kinase-1/2 levels, while the resistance to OGD was independent of the phosphatidylinositol 3'-kinase/Akt pathway. These results suggest that VEGFR-2 mediates the enhanced proliferative/OGD resistant phenotype observed in PlGF-/- astrocytesNRC publication: Ye

Gap junctions and hemichannels: communicating cell death in neurodevelopment and disease

Gap junctions are unique membrane channels that play a significant role in intercellular communication in the developing and mature central nervous system (CNS). These channels are composed of connexin proteins that oligomerize into hexamers to form connexons or hemichannels. Many different connexins are expressed in the CNS, with some specificity with regard to the cell types in which distinct connexins are found, as well as the timepoints when they are expressed in the developing and mature CNS. Both the main neuronal Cx36 and glial Cx43 play critical roles in neurodevelopment. These connexins also mediate distinct aspects of the CNS response to pathological conditions. An imbalance in the expression, translation, trafficking and turnover of connexins, as well as mutations of connexins, can impact their function in the context of cell death in neurodevelopment and disease. With the ever-increasing understanding of connexins in the brain, therapeutic strategies could be developed to target these membrane channels in various neurological disorders.Medicine, Faculty ofOther UBCNon UBCCellular and Physiological Sciences, Department ofReviewedFacult

PlGF knockout delays brain vessel growth and maturation upon systemic hypoxic challenge

In this study, we have investigated the potential role of placental growth factor (PlGF) in hypoxia-induced brain angiogenesis. To this end, PlGF wild-type (PlGF+/+) and PlGF knockout (PlGF 12/ 12) mice were exposed to whole body hypoxia (10% oxygen) for 7, 14, and 21 days. PlGF+/+ animals exhibited a significant ~40% increase in angiogenesis after 7 days of hypoxia compared with controls, while in PlGF 12/ 12 this effect only occurred after 14 days of hypoxia. No differences in pericyte/smooth muscle cell (SMC) coverage between the two genotypes were observed. After 14 days of hypoxia, PlGF 12/ 12 microvessels had a significant increase in fibrinogen accumulation and extravasation compared with those of PlGF+/+, which correlated with endothelial cell disruption of the tight junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked both pericyte/SMC coverage and endothelial vascular endothelial growth factor expression, and regressed after 21 days of hypoxia. Vascular endothelial growth factor expression levels were found to be significantly lower in the frontal cortex of PlGF 12/ 12 compared with those in PlGF+/+ animals during the first 5 days of hypoxia, which in combination with the lack of PlGF may have contributed to the delayed angiogenic response and the prothrombotic phenotype observed in the PlGF 12/ 12animals.Peer reviewed: YesNRC publication: Ye