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Targeted therapy to treat cardiovascular calcification in ESRD patients.

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of mortality for patients at all stages of chronic kidney disease (CKD) [1]. Patients with CKD are more likely to die of cardiac related health events than to even reach late stage CKD, or end-stage renal disease (ESRD) [1]. There is clinical evidence that dialysis patients with ESRD are at higher risk of developing cardiovascular disease and cardiac related mortality due to the formation of vascular calcification (VC) of the arterial walls [2]. The most severe type of VC observed in ESRD patients is that of the medial tissue layer, also known as arteriosclerosis [3]. Variations between moderate to severe arteriosclerosis is found in sixty to eighty percent of dialysis patients [4]. Arterial medial calcification directly affects the vascular smooth muscle cells (VSMCs) by calcium-phosphate mineral growth between the cells matrixes, thus hardening the medial layer which results in a loss of compliance of the artery. This arterial wall stiffening is associated with an increase in cardiac workload and elevated systolic pressure both of which are risk factors for increased cardiac morbidity and mortality [1]. VC has been regarded as a highly regulated process that occurs in a way similar to physiological bone formation [5]. In ESRD patients, the duration of dialysis treatment has been shown to negatively impact mineral metabolism, resulting in excess phosphate uptake by VSMCs [1]. In-vitro studies have shown that when exposed to calcifying media, the excess phosphate contributes to the differentiation of VSMCs into osteoblast-like cells by down regulating VSMCs contractile proteins and up-regulating bone forming proteins [1, 5]. In addition to VSMC loss of phenotype clinical studies of dialysis patients showed a negative correlation between existing VC and decreased serum concentrations of Fetuin-A, an inhibitor of extra-skeletal calcification [2,6].

Fetuin-A, also known as alpha2-Heremans-Schmid glycoprotein (AHSG) is a physiological regulator of bone metabolism [2]. This serum glycoprotein functions as a de-mineralizing agent by solubilizing, and facilitating in the removal of unwanted calcium-phosphate minerals that form as a result of calcification process [2]. In addition to Fetuin-A's inhibitory effect on VC, there are also local inhibitors of calcification that exert their function in the tissues from which they are synthesized. One such group of proteins is MGP's (matrix Gla proteins) which are vitamin K dependent and are primarily made by VSMCs and chondrocytes [7]. Vitamin K is required for the ycarboxylation of MGP, and only carboxylated MGP has the ability to act as a calcification inhibitor [1]. In-vitro calcification studies of VSMCs have shown that un-carboxylated MGP (unMGP) accumulate at sites of calcification [8]. We will use the presence of the un-carboxylated MGP at sites of calcification as a biomarker to target VC within the artery wall. Fetuin-A encapsulated by pH sensitive carriers with a surface affinity for unMGP will allow for targeted delivery of Fetuin A to sites of VC. In addition to targeted treatment of VC, intravenous injections of this therapy will reconstitute physiological levels of serum Fetuin-A possibly having a preventative effect on the further development of VC.

METHODS

VSMC Culture and Morphology. Primary human aortic vascular smooth muscle cells (ATCC, Manassas, VA) were cultured in Vascular Cell Basal Medium with 5 ng/mL rh FGH-basic, 5 mg/mL rh insulin, 50 ug/mL ascorbic acid, 10 mM L-glutamine, 5% Fetal Bovine Serum, and penicillin/streptomycin. Cell morphology was visualized using light microscope.

Immunostaining. Antibody staining was used to verify VSMC phenotype. Goat primary antibody (1:200 dilution; Pierce, Waltham, MA) a polyclonal smooth muscle a-actin is reactive for porcine and selective for the aactin found in vascular smooth muscle cells. Rabbit secondary antibody (1:200 dilution; Pierce, Waltham, MA) was tagged using a DAB substrate.

VSMC Calcification Model. Primary Human VSMCs, of passage 2 and 6, will be seeded into 4 different T75 cell culture flasks with 10 mL of growth medium, as previously described. Medium will be changed every 2 days until flasks reach confluence. Two controls will be established at this point and will continue with standard growth medium that will be replaced every 2 days for 2 weeks. Experimental flasks will be given calcification medium (standard growth medium supplemented with 10 mM p-glycerophosphate and 100 nM Dexamethasone) and medium will be changed every 2 days for 1 week. Cells will be examined regularly using a light microscope.

Quantification of Calcium Deposition. Following 1 week and 2 week time points the mineral content in the supernatant will be quantified using Calcium Assay Kit (Sigma, Waltham, MA). According to protocol, cells will be homogenized in 1.0 mL calcium assay buffer. A microcentrifuge will then be used to homogenize cell suspension at 10,000 g for 15 minutes. The supernatant will then be analyzed for calcium content using o-cresolphthalein complexosone method. The calcium content of the cell layer will be normalized to protein concentration using BCA Protein Assay Kit (Pierce, Waltham, MA).

Protein Characterization. Dynamic light scattering (DLS) was used to determine the mean diameter by number of human Fetuin-A (Sigma-Aldrich (SA); St. Louis, MO) in two different solvents, water and a KOH solution at pH 7.37 (SA). Samples were prepared by centrifuging the protein in de-ionized (DI) water solution (14.5 rpm, 5 minutes), and redispersing in the pH buffer solution to mimic physiological blood pH. Both of these solutions were analyzed using DLS. A minimum of 5 DLS measurements was collected for each sample, and averages and standard deviations for the effective diameter are reported. The molecular structure of Fetuin-A was investigated with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy using a diamond/zinc-selenide crystal (Miracle ATR[R] accessory, Pike Technology) and collecting at least 256 scans for each ATR-FTIR spectrum.

Polymersome Synthesis and Surface Topology. We will explore the use of PCL-b-PEG [poly(e-caprolactone)block- Poly(ethylene glycol)] and PMPC-b-PDPA [poly(methacryloyloxyethyl phosphorylcholine)-blockpoly(diisopropanolamine ethyl methacrylate)]; both are known pH sensitive polymersomes used in biomedical applications for drug delivery purposes [9]. Protein release will be dictated by polymersome biodegradation via hydrolysis. Two methods will be evaluated for specific recognition of the polymersome by VSMC: (1) functional group attachment to polymer before vesicle formation via ligands or moieties; and (2) post functionalization of polymersomes via covalent or strong non-covalent interactions.

RESULTS

The results from the DLS of Fetuin-A in DI water and KOH in water solution yielded a mean diameter by number of 100.57 [+ or -] 120.51 nm and 201.28 [+ or -] 66.81 nm respectively. Analysis of the ATR-FITR absorptions reveals characteristic signatures for functional groups present in Fetuin-A (Figure 1): phenol, alkene, nitrile, alkyne, ester, aromatic hydrocarbon, amine, and amine oxide.

[FIGURE 1 OMITTED]

Discussion

With respect to the DLS results, the lower standard deviation (SD) values of the mean diameter by number for the pH solution suggest a more uniform interaction of Fetuin in the pH solution compared to that of water. In order to learn more about the potential adsorption ability of Fetuin, a deeper understanding with respect to Fetuin A concentration, solvent selection, and time of interaction must be gained. Figure 1 provides insight to potential chemical bonds that can be used to tailor molecular interactions during encapsulation and targeting of the protein to effectively treat VC.

CONCLUSIONS

This targeted drug therapy has the potential to reverse and prevent further cardiac disease progression that stems from vascular calcification. With no current cure for kidney failure, and with dialysis treatments being the only alternative to kidney transplantation, this targeted therapy could effectively improve the life expectancy of patients on dialysis. The efficacy of Fetuin-A to remove newly deposited mineral content versus removing mineral deposits that have accumulated over longer periods of time has not yet been assessed. In order to determine the potential of this targeted protein delivery for both calcification therapy and preventative care we must establish an appropriate dose to ensure an effective treatment. Synthesizing the desired biodegradation profile of the polymersome after successful encapsulation of Fetuin A would be the next focus after a safe and effective dosage is found. Testing of bio-distribution, and targeted delivery will require a 3D calcification model before progression to in-vivo testing.

ACKNOWLEDGEMENTS

I would like to thank the Agricultural & Biological Engineering Department and the Swalm C. School of Chemical Engineering at Mississippi State University for providing the laboratory equipment to conduct this research. Financial support for the acquisition of the dynamic light scattering equipment was provided by EPS-0903787.

REFERENCES

[1] M. Shea and R. Holden. (2012). "Vitamin K Status and Vascular Calcification: Evidence from Observational and Clinical Studies American Society for Nutrition." Adv. Nutr. 3, pp. 158-165.

[2] P. Stenvinkel, K. Wang, A. Rahid et al. (2005, January). "Low fetuin-A levels are associated with cardiovascular death: Impact of variations in the gene encoding Fetuin." Kidney International. 67, pp. 2383-2392.

[3] A.Shioi and Y. Nishizawa. (2010). "Vascular calcification: Osteogenic transformation of vascular smooth muscle cells." J Oral Biosci. 52(1), pp. 26-32.

[4] L. J. Schurgers, E.C.M. Cranenburg, and C. Vermeer. (2008, September). "Matrix Gla -protein: The calcification inhibitor in need of vitamin K." Thromb Haemost. 100, pp. 593-603.

[5] D. Yonova. (2009). "Vascular calcification and metabolic acidosis in end stage renal disease." Hippokratia. 13(3), pp. 139-140.

[6] U.S. Department of Health and Human Services. National Kidney and Urologic Diseases Information Clearinghouse. The growing burden of kidney disease. Kidney Disease Statistics for the United States, 2012. NIH Publication No. 12-3895

[7] E. Theuwissen, E. Smit, and C. Vermeer. (2012). "The Role of Vitamin K in Soft-Tissue Calcification." American Society for Nutrition. Adv. Nutr. 3, pp.166-173.

[8] H. M. Spronk, B. A. Soute, L. J. Schurgers et al. (2001). "Matrix Gla Protein Accumulates at the Border of Regions of Calcification and Normal Tissue in the Media of the Arterial Vessel Wall." Biochemical and Biophysical Research Communications. 289, pp. 485-490.

[9] R. P. Brinkhuis, F.P.J.T. Rutjes and J. C.M. van Hest. (2011, March). "Polymeric vesicles in biomedical applications." Polymer Chemistry. 2, pp. 1449-1462.

Janice Cunningham, C. LaShan Simpson, Erick S. Vasquez, Keisha B. Walters

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Title Annotation:end-stage renal disease
Author:Cunningham, Janice; Simpson, C. LaShan; Vasquez, Erick S.; Walters, Keisha B.
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2014
Words:1676
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