K. Jane Grande-Allen

Calcific Aortic Valve Disease: Not Simply a Degenerative Process A Review and Agenda for Research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group

Calcific aortic valve disease (CAVD) encompasses the range of disease from initial alterations in the cell biology of the leaflets to end-stage calcification resulting in left ventricular outflow obstruction. The first detectable macroscopic changes in the leaflets, seen as calcification, or focal leaflet thickening with normal valve function, is termed aortic valve sclerosis, but it is likely that the initiating events in the disease process occur much earlier. Disease progression is characterized by a process of thickening of the valve leaflets and the formation of calcium nodules – often including the formation of actual bone – and new blood vessels, which are concentrated near the aortic surface. End stage disease, e.g. calcific aortic stenosis, is characterized pathologically by large nodular calcific masses within the aortic cusps that protrude through the outflow surfaces into the sinuses of Valsalva, interfering with opening of the cusps. For decades, this disease was thought to be a passive process in which the valve degenerates with age in association with calcium accumulation. Moreover, although calcific aortic valve disease is more common with age, it is not an inevitable consequence of aging. Instead, CAVD appears to be an actively regulated disease process that cannot be characterized exclusively as “senile” or “degenerative.” The NHLBI convened a group of scientists from different fields of study, including cardiac imaging, molecular biology, cardiovascular pathology, epidemiology, cell biology, endocrinology, bioengineering, and clinical outcomes, to review the scientific studies from the past decade in the field of CAVD. The purpose was to develop a consensus statement on the current state of translational research related to CAVD. Herein, we summarize recent scientific studies and define future directions for research to diagnose, treat and potentially prevent this complex disease process.

Matrix development in self-assembly of articular cartilage.

Background Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue. Methodology/Principal Findings This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks. Conclusions/Significance The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs.

BIOACTIVE POLYMER/EXTRACELLULAR MATRIX SCAFFOLDS FABRICATED WITH A FLOW PERFUSION BIOREACTOR FOR CARTILAGE TISSUE ENGINEERING

In this study, electrospun poly(ɛ-caprolactone) (PCL) microfiber scaffolds, coated with cartilaginous extracellular matrix (ECM), were fabricated by first culturing chondrocytes under dynamic conditions in a flow perfusion bioreactor and then decellularizing the cellular constructs. The decellularization procedure yielded acellular PCL/ECM composite scaffolds containing glycosaminoglycan and collagen. PCL/ECM composite scaffolds were evaluated for their ability to support the chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro using serum-free medium with or without the addition of transforming growth factor-β1 (TGF-β1). PCL/ECM composite scaffolds supported chondrogenic differentiation induced by TGF-β1 exposure, as evidenced in the up-regulation of aggrecan (11.6 ± 3.8 fold) and collagen type II (668.4 ± 317.7 fold) gene expression. The presence of cartilaginous matrix alone reduced collagen type I gene expression to levels observed with TGF-β1 treatment. Cartilaginous matrix further enhanced the effects of growth factor treatment on MSC chondrogenesis as evidenced in the higher glycosaminoglycan synthetic activity for cells cultured on PCL/ECM composite scaffolds. Therefore, flow perfusion culture of chondrocytes on electrospun microfiber scaffolds is a promising method to fabricate polymer/extracellular matrix composite scaffolds that incorporate both natural and synthetic components to provide biological signals for cartilage tissue engineering applications.

Flexural characterization of cell encapsulated PEGDA hydrogels with applications for tissue engineered heart valves.

The limitations of the current clinical options for valve replacements have inspired the development of enabling technologies to create a tissue engineered heart valve (TEHV). Poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds permit greater biological and biomechanical customization than do non-woven mesh scaffold technologies. However, the material characterization of PEGDA hydrogels has been predominantly limited to compression and tension, as opposed to bending. Since large flexural deformations result in points of maximum stress in native valves as well as TEHVs, it is crucial to evaluate any potential scaffold material in this mode. The effect of formulation parameters on the bending mechanics of cell-seeded PEGDA hydrogels were investigated with a custom designed bending tester. Three molecular weights (3.4, 6, and 8 kDa) and three weight fractions (5%, 10%, and 15%, w/v) were subjected to three-point bending tests and the flexural stiffness was calculated. Manipulating the composition of the hydrogels resulted in flexural stiffnesses comparable with native tissues (15-220 kPa) with varied mesh sizes and swelling ratios. Hydrogels containing encapsulated valve cells, methacrylated heparin (Hep-MA), or both were substantially less stiff than acellular hydrogels. In conclusion, PEGDA hydrogels are an attractive potential scaffold system for TEHVs because they are not only cytocompatible and modifiable but can also withstand bending deformations. These studies are the first to explore the encapsulation of valvular interstitial cells in pure PEGDA hydrogels as well as to investigate the bending properties of PEGDA gels.

Valve proteoglycan content and glycosaminoglycan fine structure are unique to microstructure, mechanical load and age: Relevance to an age-specific tissue-engineered heart valve.

This study characterized valve proteoglycan and glycosaminoglycan composition during development and aging. This knowledge is important for the development of age-specific tissue-engineered heart valves as well as treatments for age-specific valvulopathies. Aortic valves and mitral valves from first-third trimester, 6-week, 6-month and 6-year-old pigs were examined using immunohistochemistry for versican, biglycan, decorin and hyaluronan, as well as elastin and fibrillin. The fine structure of glycosaminoglycans was examined by fluorophore-assisted carbohydrate electrophoresis. Decorin expression was strongest in the 6-year-old valves, particularly in the aortic valve spongiosa. The quantity of iduronate was also highest in the 6-year-old valves. The central tensile-loading region of the anterior mitral leaflet demonstrated reduced glycosaminoglycan content, chain length and hydration and a larger fraction of 4-sulfated iduronate and lower fraction of 6-sulfation. With age, the anterior leaflet center showed a further increase in 4-sulfated iduronate and decrease in 6-sulfation. In contrast, the anterior leaflet free edge showed decreased iduronate and 4-sulfated glucuronate content with age. The young aortic valve was similar to the mitral valve free edge with a higher concentration of glycosaminoglycans and 6-rather than 4-sulfation, but aged to resemble the mitral anterior leaflet center, with an increase in 4-sulfated iduronate content and a decrease in the 6-sulfation fraction. Elastin and fibrillin often co-localized with the proteoglycans studied, but elastin co-localized most specifically with versican. In conclusion, composition and fine structure changes in valve proteoglycans and glycosaminoglycans with age are complex and distinct within valve type, histological layers and regions of different mechanical loading.

The Effects of Mitral Regurgitation Alone Are Sufficient for Leaflet Remodeling

Background— Although chronic mitral regurgitation results in adverse left ventricular remodeling, its effect on the mitral valve leaflets per se is unknown. In a chronic ovine model, we tested whether isolated mitral regurgitation alone was sufficient to remodel the anterior mitral leaflet. Methods and Results— Twenty-nine sheep were randomized to either control (CTRL, n=11) or experimental (HOLE, n=18) groups. In HOLE, a 2.8- to 4.8-mm diameter hole was punched in the middle scallop of the posterior mitral leaflet to create “pure” mitral regurgitation. At 12 weeks, the anterior mitral leaflet was analyzed immunohistochemically to assess markers of collagen and elastin synthesis as well as matrix metalloproteinases and proteoglycans. A semiquantitative grading scale for characteristics such as intensity and delineation of stain between layers was used to quantify differences between HOLE and CTRL specimens across the heterogeneous leaflet structure. At 12 weeks, mitral regurgitation grade was greater in HOLE versus CTRL (3.0±0.8 versus 0.4±0.4, P<0.001). In HOLE anterior mitral leaflet, saffron-staining collagen (Movat) decreased, consistent with an increase in matrix metalloproteases throughout the leaflet. Type III collagen expression was increased in the midleaflet and free edge and expression of prolyl-4-hydroxylase (indicating collagen synthesis) was increased in the spongiosa layer. The proteoglycan decorin, also involved in collagen fibrillogenesis, was increased compared with CTRL (all P≤0.05). Conclusions— In HOLE anterior mitral leaflet, the increased expression of proteins related to collagen synthesis and matrix degradation suggests active matrix turnover. These are the first observations showing that regurgitation alone can stimulate mitral leaflet remodeling. Such leaflet remodeling needs to be considered in reparative surgical techniques.

Synthesis and conformational evaluation of a novel gene delivery vector for human mesenchymal stem cells.

We have synthesized a novel gene delivery vector by covalently combining branched polyethylenimine (bPEI) and hyaluronic acid (HA) with the aim of improving transfection of bPEI into human mesenchymal stem cells (hMSCs) while maintaining cell viability. Because of the opposite charges on bPEI and HA, the bPEI-HA vector forms a zwitterionic polymer capable of inter- and intramolecular interactions. We have characterized the hydrodynamic radius of bPEI-HA and bPEI-HA/DNA complexes at ambient and physiological temperatures, as well as at a range of salt concentrations using light scattering, and investigated the effect of the size of transfecting complexes on gene delivery. We found that by increasing the salt concentration from 150 to 1000 mM of NaCl, the mean hydrodynamic radius (R(h)) of bPEI-HA increases from 2.0 +/- 1.1 to 366.0 +/- 149.0 nm. However, increasing the salt concentration decreases the mean R(h) of bPEI-HA/DNA complexes from 595.0 +/- 44.6 to 106.0 +/- 19.2 nm at 25 degrees C and from 767.0 +/- 137.2 to 74.0 +/- 23.0 nm at 37 degrees C. hMSCs transfected with smaller complexes showed a significant increase in transfection from 3.8 +/- 1.5% to 19.1 +/- 4.4%. Similarly, bPEI-HA performed significantly better than bPEI in terms of cell viability (86.0 +/- 6.7% with bPEI-HA versus 7.0 +/- 2.8% with bPEI, 24 h post exposure at the highest concentration of 500 mg/mL) and maximum transfection efficiencies (12.0 +/- 4.2% with bPEI/DNA complexes and 33.6 +/- 13.9% with bPEI-HA/DNA complexes). Thus, modifying bPEI by covalent conjugation with HA improves its performance as a gene delivery vector in hMSCs. This presents a promising approach to altering hMSCs for tissue engineering and other applications.

Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering.

The development of advanced scaffolds that recapitulate the anisotropic mechanical behavior and biological functions of the extracellular matrix in leaflets would be transformative for heart valve tissue engineering. In this study, anisotropic mechanical properties were established in poly(ethylene glycol) (PEG) hydrogels by crosslinking stripes of 3.4 kDa PEG diacrylate (PEGDA) within 20 kDa PEGDA base hydrogels using a photolithographic patterning method. Varying the stripe width and spacing resulted in a tensile elastic modulus parallel to the stripes that was 4.1-6.8 times greater than that in the perpendicular direction, comparable to the degree of anisotropy between the circumferential and radial orientations in native valve leaflets. Biomimetic PEG-peptide hydrogels were prepared by tethering the cell-adhesive peptide RGDS and incorporating the collagenase-degradable peptide PQ (GGGPQG↓IWGQGK) into the polymer network. The specific amounts of RGDS and PEG-PQ within the resulting hydrogels influenced the elongation, de novo extracellular matrix deposition and hydrogel degradation behavior of encapsulated valvular interstitial cells (VICs). In addition, the morphology and activation of VICs grown atop PEG hydrogels could be modulated by controlling the concentration or micro-patterning profile of PEG-RGDS. These results are promising for the fabrication of PEG-based hydrogels using anatomically and biologically inspired scaffold design features for heart valve tissue engineering.

Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.

Core Protein Dependence of Epimerization of Glucuronosyl Residues in Galactosaminoglycans

Chondroitin sulfate and dermatan sulfate proteoglycans are distinguished by differences in their proportion of d-glucuronosyl andl-iduronosyl residues, the latter being formed by chondroitin-glucuronate 5-epimerase during or after glycosaminoglycan chain polymerization. To investigate the influence of the core protein on the extent of epimerization, we expressed chimeric proteins in 293 HEK cells constructed from intact or modified Met1–Gln153 of decorin (DCN), which normally has a single dermatan sulfate chain at Ser34, in combination with intact or modified Leu241–Ser353 of CSF-1, which has a chondroitin sulfate attachment site at Ser309. Transfected DCNM1-Q153, like full-length DCN, contained ∼20%l-iduronate. Conversely, transfected CSF-1L241-S353, attached C-terminally on the DCN prepropeptide, contained almost exclusively d-glucuronate. Transfected intact chimeric DCNM1-Q153–CSF-1L241-S353, with two glycosaminoglycan chains, also contained almost exclusivelyd-glucuronate in chains at both sites, as did chimeras in which alanine was substituted for serine at either of the glycosaminoglycan attachment sites. Nevertheless, undersulfated intact chimeric proteoglycan was an effective substrate for epimerization of glucuronate to iduronate residues when incubated with microsomal proteins and 3′-phosphoadenylylphosphosulfate. C-terminal truncation constructs were prepared from the full-length chimera with an alanine substitution at the CSF-1 glycosaminoglycan attachment site. Transfected truncations retaining the alanine-blocked site contained chains with essentially only glucuronate, whereas those further truncated by 49 or more amino acids and missing the modified attachment site contained chains with ∼15% iduronate. This 49-amino acid region contains a 7-amino acid motif that appears to be conserved in several chondroitin sulfate proteoglycans. The results are consistent with a model in which the core protein, possibly via this motif, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase for the addition of chains with or without iduronate residues, respectively.