Daria A. Narmoneva

Injectable Self-Assembling Peptide Nanofibers Create Intramyocardial Microenvironments for Endothelial Cells

Background—Promoting survival of transplanted cells or endogenous precursors is an important goal. We hypothesized that a novel approach to promote vascularization would be to create injectable microenvironments within the myocardium that recruit endothelial cells and promote their survival and organization. Methods and Results—In this study we demonstrate that self-assembling peptides can be injected and that the resulting nanofiber microenvironments are readily detectable within the myocardium. Furthermore, the self-assembling peptide nanofiber microenvironments recruit progenitor cells that express endothelial markers, as determined by staining with isolectin and for the endothelial-specific protein platelet–endothelial cell adhesion molecule-1. Vascular smooth muscle cells are recruited to the microenvironment and appear to form functional vascular structures. After the endothelial cell population, cells that express &agr;-sarcomeric actin and the transcription factor Nkx2.5 infiltrate the peptide microenvironment. When exogenous donor green fluorescent protein–positive neonatal cardiomyocytes were injected with the self-assembling peptides, transplanted cardiomyocytes in the peptide microenvironment survived and also augmented endogenous cell recruitment. Conclusions—These experiments demonstrate that self-assembling peptides can create nanofiber microenvironments in the myocardium and that these microenvironments promote vascular cell recruitment. Because these peptide nanofibers may be modified in a variety of ways, this approach may enable injectable tissue regeneration strategies.

Endothelial cells promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration.

Background—Endothelial–cardiac myocyte (CM) interactions play a key role in regulating cardiac function, but the role of these interactions in CM survival is unknown. This study tested the hypothesis that endothelial cells (ECs) promote CM survival and enhance spatial organization in a 3-dimensional configuration. Methods and Results—Microvascular ECs and neonatal CMs were seeded on peptide hydrogels in 1 of 3 experimental configurations: CMs alone, CMs mixed with ECs (coculture), or CMs seeded on preformed EC networks (prevascularized). Capillary-like networks formed by ECs promoted marked CM reorganization along the EC structures, in contrast to limited organization of CMs cultured alone. The presence of ECs markedly inhibited CM apoptosis and necrosis at all time points. In addition, CMs on preformed EC networks resulted in significantly less CM apoptosis and necrosis compared with simultaneous EC-CM seeding (P<0.01, ANOVA). Furthermore, ECs promoted synchronized contraction of CMs as well as connexin 43 expression. Conclusions—These results provide direct evidence for a novel role of endothelium in survival and organization of nearby CMs. Successful strategies for cardiac regeneration may therefore depend on establishing functional CM–endothelium interactions.

Direct Measurement of the Poisson’s Ratio of Human Patella Cartilage in Tension

Articular cartilage has been shown to exhibit large transverse contractions when loaded in tension, suggesting the existence of large values for the Poissons ratio. Previous studies have suggested that this effect is dependent on amplitude of applied strain, so that a single Poissons ratio may not be sufficient to describe cartilage behavior. In this study, the Poissons ratio (v), toe region modulus (Eo), and linear region modulus (E) of human patellar articular cartilage were calculated in simple tension tests from optical analysis of the two-dimensional strain fields at equilibrium. The Poissons ratio was found to be independent of strain due to the absence of viscoelastic effects during testing. The Poissons ratio was found to be significantly higher in the surface zone (1.87 +/- 1.11, p<0.01) than in the middle zone (0.62 +/- 0.23), with no significant correlation of v with age of the cartilage. In general, values for Poissons ratio were greater than 0.5, suggesting cartilage behavior in tension deviates from isotropy. Reported values for the Poissons ratio of cartilage in compression have been much lower than values measured here in tension, reflecting a mechanical contribution of the collagen fibers to anisotropy in tension but not compression. The toe-region modulus (Eo) was significantly higher in the surface zone (4.51 +/- 2.78 MPa, n=8) compared to the middle zone (2.51 +/- 1.93 MPa, n=10). In addition, the linear-region modulus (E) in the surface zone, but not middle zone (3.42 +/- 2.17 MPa, n=10), was found to correlate with age (R=0.97, p<0.02) with values of surface zone E equal to 23.92 +/- 12.29 MPa (n=5) for subjects under 70 yr of age, and 4.27 +/- 2.89 MPa (n=3) for subjects over 70 yr. Moduli values and trends with depth were consistent with previous studies of human and animal cartilage. From direct measures of two independent material properties, v and E, we calculated a shear modulus, G, which had not been previously reported for cartilage from tensile testing. Calculated values for surface zone G were 3.64 +/- 1.80 MPa for subjects under 70 yr old and 0.96 +/- 0.69 MPa for subjects over 70 yr old, and were significantly higher in the surface zone than in the middle zone (1.10 +/- 0.78 MPa). This study provides an intrinsic measure for the Poissons ratio of articular cartilage and its dependence on depth which will be important in understanding the nonlinear tension-compression and anisotropic behaviors of articular cartilage.

NONUNIFORM SWELLING-INDUCED RESIDUAL STRAINS IN ARTICULAR CARTILAGE

Swelling effects in cartilage originate from an interstitial osmotic pressure generated by the presence of negatively charged proteoglycans in the tissue. This swelling pressure gives rise to a non-zero residual strain in the cartilage solid matrix in the absence of externally applied loads. Previous studies have quantified swelling effects in cartilage as volumetric or dimensional change of excised samples in varying osmotically active solutions. This study presents a new optical technique for measuring two-dimensional swelling-induced residual strain fields in planar samples of articular cartilage attached to the bone (i.e., in situ). Osmotic loading was applied to canine cartilage bone samples by equilibration in external baths of varying NaCl concentration. Non-zero swelling-induced strains were measured in physiological saline, giving evidence of the existence of residual strains in articular cartilage. Only one component of planar strain (i.e., in thickness direction) was found to be non-zero. This strain was found to be highly non-uniform in the thickness direction, with evidence of compressive strain in the deep zone of cartilage and tensile strain in the middle and surface zones. The obtained results can be used to characterize the material properties of the articular cartilage solid matrix, with estimated values of 26 M Pa for the tensile modulus for middle zone cartilage. The method provides the basis to obtain material properties of the cartilage solid matrix from a simple, free-swelling test and may be useful for quantifying changes in cartilage properties with injury, degeneration and repair.

Elastin haploinsufficiency results in progressive aortic valve malformation and latent valve disease in a mouse model.

Rationale: Elastin is a ubiquitous extracellular matrix protein that is highly organized in heart valves and arteries. Because elastic fiber abnormalities are a central feature of degenerative valve disease, we hypothesized that elastin-insufficient mice would manifest viable heart valve disease. Objective: To analyze valve structure and function in elastin-insufficient mice (Eln+/−) at neonatal, juvenile, adult, and aged adult stages. Methods and Results: At birth, histochemical analysis demonstrated normal extracellular matrix organization in contrast to the aorta. However, at juvenile and adult stages, thin elongated valves with extracellular matrix disorganization, including elastin fragment infiltration of the annulus, were observed. The valve phenotype worsened by the aged adult stage with overgrowth and proteoglycan replacement of the valve annulus. The progressive nature of elastin insufficiency was also shown by aortic mechanical testing that demonstrated incrementally abnormal tensile stiffness from juvenile to adult stages. Eln+/− mice demonstrated increased valve interstitial cell proliferation at the neonatal stage and varied valve interstitial cell activation at early and late stages. Gene expression profile analysis identified decreased transforming growth factor-&bgr;–mediated fibrogenesis signaling in Eln+/− valve tissue. Juvenile Eln+/− mice demonstrated normal valve function, but progressive valve disease (predominantly aortic regurgitation) was identified in 17% of adult and 70% of aged adult Eln+/− mice by echocardiography. Conclusions: These results identify the Eln+/− mouse as a model of latent aortic valve disease and establish a role for elastin dysregulation in valve pathogenesis.

Altered swelling behavior of femoral cartilage following joint immobilization in a canine model

Periods of reduced joint loading have been shown to induce changes in the biochemical composition, metabolism and mechanics of articular cartilage. In this study, changes in cartilage swelling behavior were studied following a 4‐week period of joint immobilization, using a recently developed osmotic loading technique [J. Biomech. 32 (1999) 401–408]. The magnitude and distribution of swelling strains were measured in cartilage–bone samples equilibrated in physiological and hypotonic saline, relative to a hypertonic reference NaCl solution. Physicochemical parameters (glycosaminoglycan fixed charge density and water volume fraction) were determined in site‐matched cartilage samples. The experimental data for swelling strains, fixed charge density and water volume fraction were used with a triphasic mechano‐chemical theory [J. Biomech. Eng. 113 (1991) 245–258] to determine the effect of joint immobilization on the tensile modulus of the cartilage solid matrix. Four weeks of immobilization resulted in a significant increase in the magnitude of swelling‐induced strains, and a significant decrease in fixed charge density in cartilage, as compared with the contralateral controls. Joint immobilization also resulted in decreases in values for the modulus of cartilage, as compared with the contralateral controls. Our results suggest that 4 weeks of joint immobilization had a significant effect on cartilage mechanical function that may be linked to collagen changes in the cartilage extracellular matrix.

REGULATION OF ENDOTHELIAL CELL ACTIVATION AND ANGIOGENESIS BY INJECTABLE PEPTIDE NANOFIBERS

RAD16-II peptide nanofibers are promising for vascular tissue engineering and were shown to enhance angiogenesis in vitro and in vivo, although the mechanism remains unknown. We hypothesized that the pro-angiogenic effect of RAD16-II results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with RAD motifs. Mouse MVECs were cultured on RAD16-II with or without integrin and MAPK/ERK pathway inhibitors, and angiogenic responses were quantified. The results were validated in vivo using a mouse diabetic wound healing model with impaired neovascularization. RAD16-II stimulated spontaneous capillary morphogenesis, and increased β(3) integrin phosphorylation and VEGF expression in MVECs. These responses were abrogated in the presence of β(3) and MAPK/ERK pathway inhibitors or on the control peptide without RAD motifs. Wide-spectrum integrin inhibitor echistatin completely abolished RAD16-II-mediated capillary morphogenesis in vitro and neovascularization and VEGF expression in the wound in vivo. The addition of the RGD motif to RAD16-II did not change nanofiber architecture or mechanical properties, but resulted in significant decrease in capillary morphogenesis. Overall, these results suggest that low-affinity non-specific interactions between cells and RAD motifs can trigger angiogenic responses via phosphorylation of β(3) integrin and MAPK/ERK pathway, indicating that low-affinity sequences can be used to functionalize biocompatible materials for the regulation of cell migration and angiogenesis, thus expanding the current pool of available motifs that can be used for such functionalization. Incorporation of RAD or similar motifs into protein engineered or hybrid peptide scaffolds may represent a novel strategy for vascular tissue engineering and will further enhance design opportunities for new scaffold materials.

Osmotic loading to determine the intrinsic material properties of guinea pig knee cartilage.

Few methods exist to study cartilage mechanics in small animal joints due to the difficulties associated with handling small tissue samples. In this study, we apply an osmotic loading method to quantify the intrinsic material properties of articular cartilage in small animal joints. Cartilage samples were studied from the femoral condyle and tibial plateau of two-month old guinea pigs. Swelling strains were measured using confocal fluorescence scanning microscopy in samples subjected to osmotic loading. A histochemical staining method was developed and calibrated for quantification of negative fixed charge density in guinea pig cartilage. Site-matched swelling strain data and fixed charge density values were then used with a triphasic theoretical model for cartilage swelling to determine the uniaxial modulus of the cartilage solid matrix. Moduli obtained in this study (7.2 MPa femoral condyle; 10.8 MPa, tibial plateau) compare well with previously reported values for the tensile moduli of human and other animal cartilages determined from uniaxial tension experiments. This study provides the first available data for material properties and fixed charge density in cartilage from the guinea pig knee and suggests a promising method for tracking changes in cartilage mechanics in small animal models of degeneration.

Regional structure–function relationships in mouse aortic valve tissue

Site-specific biomechanical properties of the aortic valve play an important role in native valve function, and alterations in these properties may reflect mechanisms of degeneration and disease. Small animals such as targeted mutagenesis mice provide a powerful approach to model human valve disease pathogenesis; however, physical mechanical testing in small animals is limited by valve tissue size. Aortic valves are comprised of highly organized extracellular matrix compartmentalized in cusp and annulus regions, which have different functions. The objective of this study was to measure regional mechanical properties of mouse aortic valve tissue using a modified micropipette aspiration technique. Aortic valves were isolated from juvenile, adult and aged adult C57BL/6 wild type mice. Tissue tensile stiffness was determined for annulus and cusp regions using a half-space punch model. Stiffness for the annulus region was significantly higher compared to the cusp region at all stages. Further, aged adult valve tissue had decreased stiffness in both the cusp and annulus. Quantitative histochemical analysis revealed a collagen-rich annulus and a proteoglycan-rich cusp at all stages. In aged adult valves, there was proteoglycan infiltration of the annulus hinge, consistent with the observed mechanical differences over time. These findings indicate that valve tissue biomechanical properties vary in wild type mice in a region-specific and age-related manner. The micropipette aspiration technique provides a promising approach for studies of valve structure and function in small animal models, such as transgenic mouse models of valve disease.

Diabetes Alters Intracellular Calcium Transients in Cardiac Endothelial Cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca2+]i) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca2+]i homeostasis due to altered sarcoplasmic reticulum Ca2+ ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca2+ regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca2+]i homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca2+]i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca2+ ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca2+]i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.