Establishment of Protein Delivery Systems Targeting Podocytes

Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes

A novel fragment derived from the β chain of human fibrinogen, β43–63, is a potent inhibitor of activated endothelial cells in vitro and in vivo

Background: Angiogenesis and haemostasis are closely linked within tumours with many haemostatic proteins regulating tumour angiogenesis. Indeed we previously identified a fragment of human fibrinogen, fibrinogen E-fragment (FgnE) with potent anti-angiogenic properties in vitro and cytotoxic effects on tumour vessels in vivo. We therefore investigated which region of FgnE was mediating vessel cytotoxicity. Methods: Human dermal microvascular endothelial cells (ECs) were used to test the efficacy of peptides derived from FgnE on proliferation, migration, differentiation, apoptosis and adhesion before testing the efficacy of an active peptide on tumour vasculature in vivo. Results: We identified a 20-amino-acid peptide derived from the β chain of FgnE, β43–63, which had no effect on EC proliferation or migration but markedly inhibited the ability of activated ECs to form tubules or to adhere to various constituents of the extracellular matrix – collagen IV, fibronectin and vitronectin. Furthermore, our data show that β43–63 interacts with ECs, in part, by binding to αvβ3, so soluble αvβ3 abrogated β43–63 inhibition of tubule formation by activated ECs. Finally, when injected into mice bearing tumour xenografts, β43–63 inhibited tumour vascularisation and induced formation of significant tumour necrosis. Conclusions: Taken together, these data suggest that β43–63 is a novel anti-tumour peptide whose anti-angiogenic effects are mediated by αvβ3

Cystatin C Deficiency Promotes Epidermal Dysplasia in K14-HPV16 Transgenic Mice

Cysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors.We tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice.Enhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization

Mechanisms and consequences of TGF-ß overexpression by podocytes in progressive podocyte disease

In patients with progressive podocyte disease, such as focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, upregulation of transforming growth factor-ß (TGF-ß) is observed in podocytes. Mechanical pressure or biomechanical strain in podocytopathies may cause overexpression of TGF-ß and angiotensin II (Ang II). Oxidative stress induced by Ang II may activate the latent TGF-ß, which then activates Smads and Ras/extracellular signal-regulated kinase (ERK) signaling pathways in podocytes. Enhanced TGF-ß activity in podocytes may lead to thickening of the glomerular basement membrane (GBM) by overproduction of GBM proteins and impaired GBM degradation in podocyte disease. It may also lead to podocyte apoptosis and detachment from the GBM, and epithelial-mesenchymal transition (EMT) of podocytes, initiating the development of glomerulosclerosis. Furthermore, activated TGF-ß/Smad signaling by podocytes may induce connective tissue growth factor and vascular endothelial growth factor overexpression, which could act as a paracrine effector mechanism on mesangial cells to stimulate mesangial matrix synthesis. In proliferative podocytopathies, such as cellular or collapsing FSGS, TGF-ß-induced ERK activation may play a role in podocyte proliferation, possibly via TGF-ß-induced EMT of podocytes. Collectively, these data bring new mechanistic insights into our understanding of the TGF-ß overexpression by podocytes in progressive podocyte disease