Krzysztof Glomski

Deletion of Adam10 in endothelial cells leads to defects in organ-specific vascular structures

During vertebrate angiogenesis, Notch regulates the cell-fate decision between vascular tip cells versus stalk cells. Canonical Notch signaling depends on sequential proteolytic events, whereby interaction of Notch with membrane-anchored ligands triggers proteolytic processing, first by Adam10 and then presenilins. This liberates the Notch intracellular domain, allowing it to enter the nucleus and activate Notch-dependent genes. Here we report that conditional inactivation of Adam10 in endothelial cells (A10ΔEC) recapitulates the increased branching and density of the retinal vasculature that is also caused by interfering with Notch signaling. Moreover, A10ΔEC mice have additional vascular abnormalities, including aberrant subcapsular hepatic veins, enlarged glomeruli, intestinal polyps containing endothelial cell masses, abnormal endochondral ossification, leading to stunted long bone growth and increased pathologic neovascularization following oxygen-induced retinopathy. Our findings support a model in which Adam10 is a crucial regulator of endothelial cell-fate decisions, most likely because of its essential role in canonical Notch signaling.

Expression profiles of Na+,K+-ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus.

During acclimation to dilute seawater, the specific activity of Na+,K+-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (< 24 h) and long (18 days) time courses. Na+,K+-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na+,K+-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na+,K+-ATPase specific activity as well as a 200% increase in Na+,K+-ATPase protein levels. Thus, it appears that the increase in Na+,K+-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the α-subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na+,K+-ATPase mRNA levels during acclimation to dilute seawater.

Intravitreal Injection of TIMP3 or the EGFR Inhibitor Erlotinib Offers Protection from Oxygen-Induced Retinopathy in Mice

PURPOSE Pathological neovascularization is a crucial component of proliferative retinopathies. Previous studies showed that inactivation of A disintegrin and metalloproteinase 17 (ADAM17), a membrane-anchored metalloproteinase that regulates epidermal growth factor receptor (EGFR) signaling, reduces pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Here, we tested how genetic inactivation of a physiological ADAM17 inhibitor, the tissue inhibitor of matrix metalloproteinases-3 (TIMP3), or intravitreal injection of TIMP3 or of the EGFR inhibitor erlotinib influenced the outcome of OIR. METHODS Wild-type mice were subjected to OIR in a chamber with 75% oxygen for 5 days beginning at postnatal day 7 (P7). Upon removal from the oxygen chamber at P12, they received a single intravitreal injection of TIMP3, erlotinib, or control. The central avascular area and neovascular tufts were measured after 5 days in room air (21% oxygen) at P17. Moreover, OIR experiments were performed with Timp3-/- mice and littermate controls. RESULTS Timp3-/- mice showed greater revascularization of the central avascular area and developed equal or fewer neovascular tufts compared to littermate controls, depending on the genetic background. Wild-type mice injected with TIMP3 or erlotinib developed fewer neovascular tufts when compared to untreated littermates. Moreover, vessel regrowth into the avascular area was reduced in TIMP3-injected mice, but not in erlotinib-injected mice. CONCLUSIONS These studies demonstrate that TIMP3 and erlotinib inhibit pathological neovascularization in the mouse retina, most likely due to inactivation of ADAM17 and the EGFR, respectively. Thus, TIMP3 and erlotinib emerge as attractive candidate antiangiogenic compounds for prevention and treatment of proliferative retinopathies.

ADAM10-Dependent Signaling Through Notch1 and Notch4 Controls Development of Organ-Specific Vascular Beds

RATIONALE Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10. OBJECTIVE The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells. METHODS AND RESULTS Mice lacking ADAM10 and ADAM17 in endothelial cells (Adam10/Adam17ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10ΔEC mice. In contrast, Adam10ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch (Adam10ΔEC(Flv)) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10ΔEC(Flv) mice than in the previously described Adam10ΔEC mice. Finally, mice lacking endothelial Notch1 (Notch1ΔEC) share some organ-specific vascular defects with Adam10ΔEC mice, whereas Notch4(-/-) mice lacking endothelial Notch1 (Notch1ΔEC/Notch4(-/-)) had defects in all vascular beds affected in Adam10ΔEC mice. CONCLUSIONS Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds.

Lack of ADAM10 in endothelial cells affects osteoclasts at the chondro-osseus junction

Mice lacking ADAM10 in endothelial cells (Adam10ΔEC mice) have shorter femurs, tibiae, and humeri than controls, raising questions about how endothelial cells could control long bone growth. We performed a histopathological evaluation of the femur and tibia growth plates at different postnatal stages, and assessed the distribution of TRAP‐positive osteoclasts and endothelial cells at the growth plate. The growth plates in Adam10ΔEC mice appeared normal at P7 and P14, but a thickened zone of hypertrophic chondrocytes and increased trabecular bone density were apparent by P21 and later. The number of TRAP+ cells at the COJ was normal at P7 and P14, but was strongly reduced at P21 and later. Moreover, the density of endomucin‐stained endothelial cells at the COJ was increased starting at P7. The defects in long bone growth in Adam10ΔEC mice could be caused by a lack of osteoclastogenesis at the COJ. Moreover, ADAM10 appears to regulate endothelial cell organization in the developing bone vasculature, perhaps in a similar manner as in the developing retinal vascular tree, where ADAM10 is thought to control Notch‐dependent endothelial cell fate decisions. This study provides evidence for the regulation of osteoclast function by endothelial cells in vivo.

Case 28-2017. A 13-Month-Old Girl with Pneumonia and a 33-Year-Old Woman with Hip Pain.

A 13-month-old girl presented with pneumonia. Imaging studies revealed splenomegaly, splenic lesions, and diffuse lymphadenopathy. Three months later, her mother presented with right hip pain and a lytic lesion in the femoral neck. Diagnoses were made.

Metastatic Follicular Thyroid Carcinoma and the Primary Thyroid Gross Examination: Institutional Review of Cases from 1990 to 2015

The diagnosis of follicular-patterned carcinomas, including follicular thyroid carcinoma, oncocytic (Hürthle cell) carcinoma, and the encapsulated follicular variant of papillary thyroid carcinoma, requires evidence of capsular and/or vascular invasion. With minimally invasive carcinomas classified often within less than a millimeter of tissue segregating them from adenomas and non-invasive follicular thyroid neoplasms with papillary-like nuclear features, opinions vary internationally over how much of the capsule to submit in order to deem it well enough represented, considering that even if grossly entirely submitted in microcassettes, without leveling through each tissue block, the capsule is truly never entirely examined microscopically. Here, we retrospectively examine submission practices and outcomes at a single, high-volume institution over a 25-year period. Our results indicate that the vast majority of lesions with poor outcomes are those with wide invasion, and tumors lacking gross evidence of capsular perturbation rarely lead to recurrence or metastasis, an unsurprising result that should prompt re-evaluation of our grossing methods and approach to follicular-patterned tumors in a time of cost restraint, molecular diagnostics, and low biological potential of encapsulated, circumscribed neoplasia of the thyroid.