Endothelial Cell Targeting by cRGD-Functionalized Polymeric Nanoparticles under Static and Flow Conditions.

Since αvß3 integrin is a key component of angiogenesis in health and disease, Arg-Gly-Asp (RGD) peptide-functionalized nanocarriers have been investigated as vehicles for targeted delivery of drugs to the αvß3 integrin-overexpressing neovasculature of tumors. In this work, PEGylated nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA) functionalized with cyclic-RGD (cRGD), were evaluated as nanocarriers for the targeting of angiogenic endothelium. For this purpose, NPs (~300 nm) functionalized with cRGD with different surface densities were prepared by maleimide-thiol chemistry and their interactions with human umbilical vein endothelial cells (HUVECs) were evaluated under different conditions using flow cytometry and microscopy. The cell association of cRGD-NPs under static conditions was time-, concentration- and cRGD density-dependent. The interactions between HUVECs and cRGD-NPs dispersed in cell culture medium under flow conditions were also time- and cRGD density-dependent. When washed red blood cells (RBCs) were added to the medium, a 3 to 8-fold increase in NPs association to HUVECs was observed. Moreover, experiments conducted under flow in the presence of RBC at physiologic hematocrit and shear rate, are a step forward in the prediction of in vivo cell-particle association. This approach has the potential to assist development and high-throughput screening of new endothelium-targeted nanocarriers.

Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.

Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form "pre-micrometastases," in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation.

Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells.

Angiogenic endothelium plays a crucial role in tumor growth. During angiogenesis, complex alterations in the microenvironment occur. In response, the endothelium undergoes phenotypic changes, for example overexpression of alpha(v)-integrins. Here, we show that the overexpression of alpha(v)-integrins on angiogenic endothelial cells is engaged in phagocytic actions involving binding ("tethering") and uptake ("tickling") of lactadherin (also termed MFG-E8)--opsonized particles. Phosphatidylserine (PS)--exposing multilamellar vesicles, "aged" erythrocytes, and apoptotic melanoma cells incubated with lactadherin were all phagocytosed by angiogenic endothelial cells in vitro. Furthermore, we demonstrated lactadherin expression in and around tumor blood vessels making opsonization in situ plausible. By engineering the surface of erythrocytes with covalently coupled cyclic Arg-Gly-Asp (RGD) peptides--mimicking lactadherin opsonization--we could induce phagocytosis by angiogenic endothelial cells both in vitro and in vivo. In vitro, this was confirmed by cytochalasin D preincubation. When RGD-erythrocytes were administered intravenously in tumor-bearing mice, blood vessel congestion followed by tumor core necrosis was seen. Moreover, RGD-erythrocytes could delay tumor growth in a murine melanoma model, possibly through induction of tumor infarctions. These results reveal that angiogenic endothelial cells have phagocytic properties for lactadherin-opsonized large particles and apoptotic cells. Implications of our findings for diagnostic and therapy of angiogenesis-driven diseases are discussed.

Rab3D and actin reveal distinct lamellar body subpopulations in alveolar epithelial type II cells.

Rab3D is a small GTP-binding protein associated with secretory vesicles in various exocrine and endocrine cells, where it has been implicated in regulated exocytosis. Data obtained previously in pancreas have suggested that rab3D is involved in the coating of secretory granules with filamentous actin. In the present study we employed Western blot analysis, immunofluorescence, and immunoelectron microscopy to examine the distribution of rab3D in rat lung. Rab3D immunoreactivity was detected in bronchiolar Clara cells and alveolar epithelial type II (AET-II) cells. In both cell types, rab3D displayed preferential localization to secretory vesicles that were identified using specific antibodies against Clara Cell Secretory Protein and p180 lamellar body protein, respectively. Interestingly, rab3D was associated with only 24% of the lamellar bodies in AET-II cells. Rab3D-positive lamellar bodies were typically in close proximity of the apical plasma membrane, where exocytosis occurs. Another subpopulation of lamellar bodies, constituting only 2%, was not only rab3D-positive but could also be labeled with the filamentous-actin probe phalloidin. A third subpopulation, constituting 9%, displayed actin coating without rab3D staining. We propose that these three lamellar body subpopulations represent consecutive intermediates along the regulated exocytotic pathway, implying that rab3D release and actin coating are intimately linked processes.

Water distribution and related morphology in human stratum corneum at different hydration levels.

This study focused on the water distribution in human stratum corneum and on the swelling of the corneocytes. For this purpose stratum corneum was hydrated to various levels and used either for Fourier transform infrared spectroscopy or for cryo-scanning electron microscopy. The images were analyzed with respect to water localization and cell shape. The Fourier transform infrared spectra were measured to study the water-lipid interactions. The results show that water only slightly changes the lipid transitions in the stratum corneum even at a hydration level of 300% wt/wt compared to stratum corneum and that water is inhomogeneously distributed in the stratum corneum. No gradual increase in water level was observed in depth. At 57%-87% wt/wt water content the hydration level in the central part of stratum corneum is higher than in the superficial and deeper cell layers. Water domains are mainly present within the corneocytes and not in the intercellular regions. At a very high hydration level (300% wt/wt), the corneocytes are strongly swollen except for the deepest cell layers adjacent to the viable epidermis. The corneocytes in these layers are not swollen. At 300% wt/wt hydration level water domains are also present in intercellular regions. Between 17% wt/wt and 300% wt/wt the cell thickness increases linearly with the hydration level suggesting that swelling of cells mainly occurs in the direction perpendicular to the skin surface. At an increased hydration level, the corneocyte envelope more efficiently surrounds the cell content compensating for the increased cell volume. The changes in stratum corneum morphology with increasing water level have also been observed in dermatomed skin.