DESIGN AND CHARACTERIZATION OF
ARTIFICIAL EXTRACELLULAR MATRIX PROTEINS FOR USE AS SMALL-DIAMETER VASCULAR GRAFTS
Thesis by Sarah Heilshorn
In Partial Fulfillment of the Requirements for the degree of
Doctor of Philosophy
CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California
2004
(Defended May 10, 2004)
„ 2004 Sarah Heilshorn All Rights Reserved
ACKNOWLEDGMENTS
First, I thank my thesis advisor, David Tirrell, for his guidance and support. He generously shared with me his scientific insight, strong sense of purpose and integrity, and thoughtful counsel. Our many interesting discussions, about matters both scientific and otherwise, have helped me immensely in my scientific and personal development.
I would also like to thank my thesis committee, Anand Asthagiri, Mark Davis, and Barbara Wold, for their valuable advice and comments. I also thank Profs. Tetsuji Yamaoka and Rob Phillips for providing me with personal guidance and scientific enthusiasm. As I embark on my own scientific career, I am fortunate to have such excellent role models.
The many members of the Tirrell Group, past and present, have been wonderful colleagues. Our daily interactions made life inside and outside of the lab enjoyable. Thank you all for your friendship and scientific creativity.
Several members of the Caltech community have provided me with continous support and understanding. Thank you to the WEST steering committee, the supportive Caltech staff, and the faculty who have taken time to participate in our activities. My interactions with all of you have greatly enhanced my graduate school experience.
Thank you to my friends, both at Caltech and around the world. Your encouragement has kept me both focused and balanced.
My family has been the foundation of my success. Their never-failing love and faith in me has given me the confidence to pursue my dreams.
Finally, thank you to my fiancé, Andrew Spakowitz, for his friendship, love, honesty, and trust.
ABSTRACT
Design and Characterization of Artificial Extracellular Matrix Proteins for Use as Small-Diameter Vascular Grafts
May 2004
Sarah Heilshorn, B.S., Georgia Institute of Technology M.S., California Institute of Technology
Ph.D. California Institute of Technology
Synthetic small-diameter vascular grafts often fail within three years of implantation.
The underlying causes of graft failure are thought to be i) a mismatch in the mechanical properties between the graft and host material and ii) an inability of the graft to support the adhesion of endothelial cells. To address these two issues, artificial extracellular matrix (aECM) proteins contain elastin-like regions to provide physical integrity and cell-binding domains derived from fibronectin to promote endothelial cell attachment. Using recombinant protein technology, a family of artificial proteins was created with differing ratios of elastin-like regions to cell-binding domains, with variable placement of amino acid crosslinking residues, and with differing identity of cell-binding domain.
Human umbilical vein endothelial cells (HUVEC) adhere in a sequence-specific manner to aECM proteins, secrete basal levels of key fibrinolytic regulators, and are capable of resisting a physiologically relevant detachment force. HUVEC spread more quickly and adhere more firmly to aECM proteins that contain the RGD versus the CS5 cell-binding domain. Decreasing the density of RGD cell-binding domains results in
decreased HUVEC adhesion. Furthermore, amino acid selection even at sites up to 16 residues away from the cell-binding domain impacts HUVEC spreading and adhesion.
HUVEC also adhere more strongly to stiffer aECM films. Therefore, the identity, density, and context of the cell-binding domain as well as the elastic modulus of the substrate are all important variables in influencing cell-substrate interactions.
Proper amino acid sequence choice also influences the susceptibility of aECM proteins to elastase proteolysis; modifying 3% of the amino acid side chains results in a 7- fold reduction in degradation rate. An alternative strategy to decrease degradation involves incorporation of the noncanonical amino acid, 5,5,5-trifluoroisoleucine, into the favored proteolytic cut site, isoleucine. Replacing 82% of the isoleucines results in a twofold reduction in degradation rate without compromising sequence-specific HUVEC adhesion.
Incorporation of another noncanonical amino acid, para-azidophenylalanine, allows synthesis of photoreactive proteins that can be patterned using photolithography. These protein patterns retain their ability to adhere HUVEC and produce stable cell patterns after 48 hours in medium supplemented with serum.
TABLE OF CONTENTS
Acknowledgments...iii
Abstract ... iv
Table of Contents... vi
List of Tables... xi
List of Figures ... xii
Abbreviations ... xiv Chapter
I: Introduction ...I-1 1. Protein engineering approach to biomaterials ...I-1 2. Small-diameter vascular grafts...I-2 3. Artificial extracellular matrix proteins - Design strategy...I-4 4. Artificial extracellular matrix proteins - Previous results ...I-6 5. Artificial extracellular matrix proteins - Current results...I-7 6. References...I-9 II: Endothelial cell adhesion to the fibronectin CS5 domain in artificial
extracellular matrix proteins... II-1 1. Introduction... II-2 2. Materials and methods... II-5 2.1. Protein expression and purification... II-5 2.2. Lipopolysaccharide quantitation ... II-6 2.3. Cell maintenance... II-6 2.4. Substrate preparation and characterization ... II-6 2.5. Phase contrast density slicing... II-7 2.6. Comparison and inhibition of endothelial cell
binding to aECM proteins... II-7 2.7. Cell detachment by shear stress... II-8 2.8. Secretion of tPA and PAI-1... II-10
3. Results... II-10 3.1. Synthesis and analysis of aECM proteins... II-10 3.2. HUVEC adhesion to aECM proteins ... II-10 3.3. HUVEC adhesion under physiologically relevant
shear stresses ... II-12 3.4. Secretion of fibrinolytic regulators ... II-13 4. Discussion ... II-15 5. Conclusion and acknowledgments... II-18 6. References... II-18 III: Comparative cell response to artificial extracellular matrix proteins
containing the RGD and CS5 cell-binding domains...III-1 1. Introduction...III-2 2. Materials and methods...III-4 2.1. Protein expression and purification...III-4 2.2. Cell culture...III-5 2.3. Surface preparation...III-5 2.4. Peptide inhibition...III-6 2.5. Cell adhesion...III-6 2.6. Cell spreading ...III-7 2.7. Immunofluorescence microscopy ...III-8 3. Results and discussion ...III-9 3.1. Competitive peptide inhibition...III-9 3.2. HUVEC resistance to detachment forces...III-10 3.3. Time course of HUVEC spreading...III-12 3.4. Visualization of focal adhesions and integrin clusters ...III-13 4. Conclusion and acknowledgments...III-17 5. References...III-18
IV: Cell-binding domain context affects cell behavior on engineered
proteins ...IV-1 1. Introduction...IV-2 2. Experimental section ...IV-5 2.1. Protein expression and purification...IV-5 2.2. Protein adsorption...IV-5 2.3. Cell culture...IV-5 2.4. Cell spreading – Scanning electron microscopy...IV-5 2.6. Cell spreading – Phase contrast microscopy...IV-6 2.7. Cell resistance to detachment ...IV-6 3. Results and discussion ...IV-6 3.1. Protein synthesis and characterization ...IV-6 3.2. HUVEC resistance to detachment forces...IV-7 3.3. HUVEC spreading on engineered proteins...IV-10 4. Conclusion and acknowledgments...IV-12 5. Supporting information ...IV-13 6. References...IV-14 V: Controlled enzymatic degradation of artificial proteins engineered
for cell adhesion ...V-1 1. Introduction...V-2 2. Experimental section ...V-5 2.1. Protein expression and purification...V-5 2.2. Analysis of elastase degradation fragments...V-5 2.3. Kinetics of elastase degradation...V-6 2.4. Crosslinking of protein films...V-6 2.5. Elastase degradation of crosslinked films...V-7 2.6. Weight fraction protein...V-7 3. Results and discussion ...V-7 3.1. Protein expression and purification...V-7
3.2. Elastase degradation of engineered proteins with
altered elastin-like sequences ...V-8 3.3. Elastase degradation of crosslinked protein films ...V-13 4. Conclusion and acknowledgments...V-17 5. References...V-17 VI: Controlled degradation of a cell-adhesive, elastomeric protein
through incorporation of a fluorinated amino acid...VI-1 1. Introduction...VI-2 2. Results and discussion ...VI-4 2.1. Protein synthesis and characterization ...VI-4 2.2. Protein degradation...VI-7 2.3. Endothelial cell adhesion...VI-10 3. Methods...VI-12 3.1. Protein expression and purification...VI-12 3.2. Tryptic digest/MALDI ...VI-12 3.3. LCST measurement...VI-13 3.4. Analysis of elastase degradation ...VI-13 3.5. Cell adhesion...VI-14 4. Supporting information ...VI-15 5. References...VI-15 VII: Lithographic patterning of an intrinsically photoreactive cell-
adhesive protein ... VII-1 1. Introduction, results, and discussion... VII-2 2. References and notes ... VII-11 VIII: Coordinated regulation of cell adhesion by cell-binding domain
density and elastic modulus ...VIII-1 1. Introduction...VIII-2 2. Experimental section ...VIII-4
2.1. Protein expression and purifcation...VIII-4 2.2. Protein crosslinking ...VIII-4 2.3. Cell culture...VIII-5 2.4. Cell resistance to detachment ...VIII-5 2.5. Zymography...VIII-5 3. Results and discussion ...VIII-6 3.1. Sequence-specific cell adhesion...VIII-6 3.2. Tuning cell adhesion through material properties ...VIII-8 3.3. Matrix metalloproteinase-2 activation ...VIII-10 4. Conclusion ...VIII-12 5. References...VIII-12 IX. Conclusion ...IX-1 1. Summary...IX-1 2. Future research directions ...IX-2 3. References...IX-3 Appendix A: Symbols for Amino Acids...A-1 Appendix B: Detailed Experimental Protocols... B-1 Appendix C: Cloning Schemes and Plasmid Maps ... C-1
LIST OF TABLES
Number Page
II-1. Secretion levels of tPA and PAI-1 by HUVEC monolayers on
various substrates ... II-14 V-1. N-terminal sequencing results of selected proteolytic fragments ...V-10 V-2. Physical properties and degradation rates of films of crosslinked
elastin-like sequences...V-14 VIII-1. MMP-2 secretion and activation by HUVEC monolayers grown on
various aECM films...VIII-11
LIST OF FIGURES
Number Page
II-1. Amino acid sequences of aECM proteins... II-3 II-2. HUVEC adhesion to aECM 1 and 2 ... II-11 II-3. Peptide inhibition of HUVEC adhesion on aECM 3... II-12 II-4. HUVEC resistance to detachment under shear stress... II-13 II-5. PAI-1 secretion over 72 hours... II-15 III-1. Amino acid sequences of aECM proteins...III-4 III-2. Competitive peptide inhibition...III-10 III-3. HUVEC resistance to detachment forces...III-11 III-4. Cell spreading on aECM substrates...III-13 III-5. Fluorescence micrographs of actin filaments and
focal adhesions in HUVEC on aECM proteins ...III-14 III-6. Fluorescence micrographs of actin filaments and integrins ...III-16 IV-1. Amino acid sequences of aECM proteins...IV-4 IV-2. Adsorption isotherms of engineered proteins ...IV-7 IV-3. Cell resistance to detachment forces on various aECM
concentrations ...IV-9 IV-4. HUVEC resistance to normal detachment forces ...IV-10 IV-5. Scanning electron micrographs of HUVEC on Ki3 and Kt3 ...IV-11 IV-6. Percent of spread HUVEC on engineered proteins ...IV-11 IV-S1. Cell resistance to normal detachment forces on glass ...IV-13 IV-S2. Western analysis of histidine- and T7-tag cleavage ...IV-13 IV-S3. Cell resistance to detachment on proteins with tags removed...IV-14 V-1. Amino acid sequences of aECM proteins...V-5 V-2. SDS-PAGE of elastase degradation fragments...V-9 V-3. Preferred elastase cleavage sites...V-10 V-4. Western blot of full-length, intact proteins...V-11 V-5. Full-length protein remaining after HLE exposure ...V-11
V-6. Kinetic analysis of elastase degradation...V-12 V-7. Decrease in elastic modulus after HLE exposure, low moduli ...V-15 V-8. Decrease in elastic modulus after HLE exposure, high moduli...V-16 VI-1. Amino acid sequences of aECM proteins...VI-3 VI-2. MALDI-TOF of tryptic digest fragments ...VI-6 VI-3. LCST analysis...VI-7 VI-4. Number of new N-termini created by HLE degradation...VI-8 VI-5. Western-blot of full-length intact proteins...VI-9 VI-6. HUVEC adhesion to engineered proteins ...VI-10 VI-S1. Kinetic analysis of HLE degradation ...VI-15 VII-1. Design and production of aECM-N3... VII-4 VII-2. Characterization of azide decomposition... VII-6 VII-3. Optical and physical characterization of photopatterned films... VII-8 VII-4. Fluorescence microscopy of photopatterned films... VII-9 VII-5. HUVEC attachment to photopatterned films... VII-10 VIII-1. Amino acid sequences of aECM proteins...VIII-4 VIII-2. HUVEC adhesion to crosslinked RGD proteins...VIII-6 VIII-3. Sequence-specific HUVEC adhesion to RGD proteins...VIII-7 VIII-4. HUVEC adhesion to crosslinked REDV proteins ...VIII-8 VIII-5. HUVEC adhesion in response to RGD cell-binding domain
density ...VIII-9 VIII-6. HUVEC adhesion in response to elastic modulus ...VIII-10 VIII-7. Gelatin zymogram of conditioned medium samples...VIII-11
ABBREVIATIONS
5TFI 5,5,5-trifluoroisoleucine
aECM Artificial extracellular matrix protein
BCA Bicinchoninic acid
BS3 Bis(sulfosuccinimidyl) suberate
BSA Bovine serum albumin
CAI Cell adhesion index
DPN Dip-pen nanolithography
E Elastic modulus
ECM Extracellular matrix
ePTFE expanded poly(tetrafluoroethylene) ELISA Enzyme-linked immunosorbent assay
HLE Human leukocyte elastase
HMDI Hexamethylene diisocyanate
HUVEC Human umbilical vein endothelial cells LAL Limulus amebocyte lysate gel-clot assay LCST Lower critical solution temperature
LPS Lipopolysaccharide
MALDI-MS Matrix-assisted laser desorption ionization-mass spectrometry
Mc Molecular weight between crosslinks
PAI-1 Plasminogen activator inhibitor
PBS Phosphate buffered saline
PET Poly(ethylene terephthalate)
pN3Phe Para-azidophenylalanine
SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electrophoresis
SEM Scanning electron microscopy
tPA Tissue plasminogen activator
TSAT Tri-succinimidyl aminotriacetate
Note: Single letter abbreviations for the natural amino acids are listed in Appendix A.
