Cardiac involvement in mixed connective tissue disease: A systematic review

Objective

To report the clinical characteristic of cardiac disease in patients with mixed connective tissue disease (MCTD).

Method

We identified published case series that reported cardiac manifestations of patients with MCTD by searching the PubMed database using the search terms “mixed connective tissue disease”. We identified 11 case series that met our eligibility criteria.

Result

616 patients were included. Prevalence of cardiac involvement varied from 13% to 65% depending on patient selection and method used for detection. Pericarditis was the most common cardiac diagnosis with a prevalence of 30% and 43% in two prospective studies. Non-invasive cardiac tests, including electrocardiogram and echocardiogram, detected subclinical cardiac abnormalities in 6%–38% of patients. These abnormalities included conduction abnormalities, pericardial effusion and mitral valve prolapse. Diastolic dysfunction and accelerated atherosclerosis were well-documented in a case–control study. Three prospective studies revealed an overall mortality of 10.4% over the period of follow-up of 13–15 years. 20% of the mortality was directly attributable to cardiac cause.

Conclusion

Cardiac involvement was common among patients with MCTD though the involvement was often clinically inapparent. Non-invasive cardiac tests might have a role for subclinical disease screening for early diagnosis and timely treatment as cardiac involvement was one of the leading causes of mortality.     

Explaining survival differences between two consecutive studies with allogeneic stem cell transplantation in patients with chronic myeloid leukemia

Purpose

In the two consecutive German studies III and IIIA on chronic myeloid leukemia, between 1995 and 2004, 781 patients were randomized to receive either allogeneic hematopoietic stem cell transplantation with a related donor or continued drug treatment. Despite comparable transplantation protocols and most centers participating in both studies, the post-transplant survival probabilities for patients transplanted in first chronic phase were significantly higher in study IIIA (144 patients) than in study III (113 patients). Prior to the decision on a combined analysis of both studies, reasons for this discrepancy had to be investigated.

Methods

The Cox proportional hazard cure model was used to identify prognostic factors for post-transplant survival.

Results

Donor–recipient matching for human leukocyte antigen, patient age, time between diagnosis and transplantation, and calendar time showed a significant influence on survival and/or the incidence of cure. Added as a further factor, affiliation to study IIIA had no significant impact any longer.

Conclusions

Discrepancies in influential prognostic factors explained the different post-transplant survival probabilities between the studies. The significance of calendar time suggests a lack of consistency of transplantation practice over time. Accordingly, the prerequisite for a common assessment of overall survival in the two randomized transplantation arms was not met. Moreover, our analyses provide an independent validation of established prognostic factors and their cutoffs. The statistical approach in investigating and modeling potential prognostic factors for survival sets an example for the examination of studies with unexpected outcome differences in concurrent treatment arms.     

Impact of moderate calorie restriction on testicular morphology and endocrine function in adult rhesus macaques (Macaca mulatta)

We previously reported that moderate calorie restriction (CR) has minimal impact on testicular gene expression in young adult rhesus macaques, and no obvious negative impact on semen quality or plasma testosterone levels. We now extend these findings by examining the influence of CR on various aspects of the reproductive axis of older males, including 24-h circulating testosterone levels, testicular gene expression, and testicular morphology. Young adult and old adult male rhesus macaques were subjected to either 30 % CR for 5–7 years, or were fed a standard control diet. Analysis of the 24-h plasma testosterone profiles revealed a significant age-associated decline, but no evidence for CR-induced suppression in either the young or old males. Similarly, expression profiling of key genes associated with testosterone biosynthesis and Leydig cell maintenance showed no significant CR-induced changes in either the young or old animals. The only evidence for CR-associated negative effects on the testis was detected in the old animals at the histological level; when old CR animals were compared with their age-matched controls, there was a modest decrease in seminiferous tubule diameter and epithelium height, with a concomitant increase in the number of depleted germ cell lines. Reassuringly, data from this study and our previous study suggest that moderate CR does not negatively impact 24-h plasma testosterone profiles or testicular gene expression. Although there appear to be some minor CR-induced effects on testicular morphology in old animals, it is unclear if these would significantly compromise fertility.     

Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking

The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.Regenerative medicine and tissue engineering are providing new avenues to treat a multitude of conditions and diseases through restoring, maintaining, or enhancing the function of a wide range of tissues (e.g., muscle, bone, skin) and organs (e.g., heart, lungs, kidneys). Despite recent advancements, the clinical utility of many engineered tissues remains dependent upon the development of methods that promote mechanically robust tissues capable of withstanding the in vivo environment. This problem presents a critical hurdle for efforts geared toward developing de novo musculoskeletal tissues, such as cartilage, meniscus, tendons, and ligaments, for reparative and regenerative strategies. Tissue engineering has the potential to provide alternative treatment options for musculoskeletal injury and disease by generating neotissue that mimics the complex structure of native tissue (1, 2). The objective is to improve the properties of the engineered tissues to achieve native tissue biomechanical properties, maturation, and long-term functionality. For example, in articular cartilage tissue engineering, a variety of methods, including combinations of hydrostatic pressure and growth factor treatment (3), fluid flow (4), and matrix-modulating enzymes (5), have resulted in improved neotissues with tensile moduli ranging from 1.3 to 2.3 MPa; however, native tissue values range from 5 to 25 MPa (6). Similarly, in neofibrocartilage, tensile properties have been reported to be 2.5–3.5 MPa (7, 8), with native fibrocartilage values ranging from 7 to 295 MPa (9, 10). These low properties likely result from the immaturity of the ECM of the neotissues, which is mainly due to lack of collagen cross-links (11). Therefore, it is important that additional treatment modalities be evaluated toward enhancing neotissue tensile properties.Collagen, the most prevalent fibrous protein in the body, supports the mechanical integrity of tissues and organs. In musculoskeletal tissues, collagen comprises the major fraction of the ECM and accounts for ∼65–80% of these tissues’ dry weights (12). After collagen biosynthesis and triple-helix formation, the copper-dependent enzyme lysyl oxidase (LOX) catalyzes extracellular modification of lysine and hydroxylysine amino acids of the collagen fibers into their aldehyde forms (Fig. 1A). Sequentially, this modification induces the formation of covalent pyridinoline (PYR) cross-links between individual collagen fibers (13) (Fig. 1B) and stabilizes the formation of heterotypic fibrils (Fig. 1C) and, subsequently, the tissue ECM. These intermolecular collagen cross-links, present in all native musculoskeletal tissues, signify the maturity of the tissue’s ECM (14). In particular, cartilaginous tissues, ligaments, and tendons feature two forms of collagen cross-links, the difunctional (initial/immature) cross-link dehydrodihydroxylysinonorleucine and the trifunctional (mature) cross-link hydroxylysyl-PYR (15) (Fig. 1B). Several studies have highlighted the pivotal role of these cross-links in native tissues’ biomechanical or functional properties (11, 16, 17). For example, in cartilage, the cross-linked collagen fibrils noncovalently stabilize the highly hydrated, negatively charged proteoglycans (18). These findings suggest the need for recapitulating cross-linking in engineered collagen-rich tissues.Open in a separate windowFig. 1.This illustration represents a hierarchical depiction of a heterotypic collagen fibril that is commonly found in most musculoskeletal tissues, such as cartilage, tendons, and ligaments. The figure emphasizes the internal axial relationships required for the formation of mature cross-links. (A) Detailed view of the axial stagger of individual collagen molecules required for PYR cross-linking. (B) Illustration of relationships among neighboring axial fibers of trifunctional (mature) hydroxylysyl-PYR collagen cross-links. (C) Illustration of a 3D concept of a heterotypic fibril, commonly found in musculoskeletal tissues, consisting of collagen types II (yellow), IX (red), and XI (blue). Figure adapted from ref. 68.Only a few studies to date have evaluated the extent of collagen cross-links (19, 20) and, more importantly, their influence on the tensile and compressive properties of engineered musculoskeletal tissues. In contrast, many studies have investigated how other ECM components, such as glycosaminoglycan (GAG), collagen, and mineralization, contribute to biomechanical properties (19, 21–23). Such work has yielded variable results. Specifically, although it has been suggested that the compressive properties of native articular cartilage are best correlated with the proteoglycan component of the ECM (24), more recent studies have shown a better correlation of the compressive modulus with a combination of both GAG and collagen content (25–27). For engineered tissues, tensile stiffness has been strongly associated with total collagen content (22) as well as PYR content (28). Nevertheless, a significant correlation with compressive properties has also been reported with respect to the PYR content of engineered tissues (28). These findings might suggest that the various tissue biomechanical properties are dependent on more than just the quantity of specific biochemical components (29). Therefore, the unique structural organization, cross-linking, and architecture of the collagen network also likely play equally important roles, along with collagen and GAG quantities, in determining the biomechanical functionality of the tissue.To the best of our knowledge, there are no validated and optimized methods for promoting collagen cross-linking and concomitant improvement in biomechanical properties in a spectrum of native and engineered tissues. Low oxygen tension, however, has been shown to have various molecular effects in different tissues (30, 31) through a hypoxia-dependent mechanism. This molecular mechanism is based on the critical involvement of hypoxia-inducible factor-1 (HIF-1) (32), which is present in many cells (33–35), including articular chondrocytes (36). In diarthrodial joints as well as developmental growth plates, chondrocytes experience hypoxic conditions (37). Low oxygen tension has been shown to affect the metabolism of articular chondrocytes, targeting the production of tissue-specific cartilage ECM proteins (38–42). Deletion of HIF-1α results in chondrocyte death, along with diminished expression of the cyclin-dependent kinase inhibitor p57, highlighting the importance of HIF-1 regulation for cell survival and growth arrest in this hypoxic environment (43). The critical role of HIF-1 in the chondrogenic differentiation of rat mesenchymal stem cells as well as human ES cells has been also identified (42, 44–46). Hypoxia-induced HIF-1 stabilization has been shown to control LOX regulation (47, 48), suggesting that hypoxia could affect ECM stabilization and tissue maturation through LOX-induced PYR cross-link formation. The manifold role of HIF-1 raises the intriguing idea that the ECM of native and engineered tissues and, concomitantly, their functional properties could be manipulated through hypoxia-mediated stimulation of HIF-1–regulated pathways, LOX gene expression, and sequential collagen cross-link formation.Although studies have elucidated the pathway underlying the effects of hypoxia on cartilage growth, function, and synthesis, the ability of endogenous (hypoxia-induced) and exogenous LOX application to enhance collagen cross-linking and concomitant mechanical properties in both native and engineered tissues remains unexplored and is the central hypothesis of the present study. In the series of studies presented here, a variety of musculoskeletal tissues widely used in clinical practice as grafts to replace degenerative tissues as well as neocartilage constructs were investigated to assess hypoxia- and LOX-mediated collagen cross-linking manipulation. The overall goal was to preserve and promote the functional properties of the native explants and neotissues, respectively. Strategically targeting collagen cross-links may be useful for engineering tissues de novo, for elucidating disease processes, and for the development of potential novel therapeutic modalities.     

Mechanotransduction of fluid stresses governs 3D cell migration

Solid tumors are characterized by high interstitial fluid pressure, which drives fluid efflux from the tumor core. Tumor-associated interstitial flow (IF) at a rate of ∼3 µm/s has been shown to induce cell migration in the upstream direction (rheotaxis). However, the molecular biophysical mechanism that underlies upstream cell polarization and rheotaxis remains unclear. We developed a microfluidic platform to investigate the effects of IF fluid stresses imparted on cells embedded within a collagen type I hydrogel, and we demonstrate that IF stresses result in a transcellular gradient in β1-integrin activation with vinculin, focal adhesion kinase (FAK), FAK^PY397, F actin, and paxillin-dependent protrusion formation localizing to the upstream side of the cell, where matrix adhesions are under maximum tension. This previously unknown mechanism is the result of a force balance between fluid drag on the cell and matrix adhesion tension and is therefore a fundamental, but previously unknown, stimulus for directing cell movement within porous extracellular matrix.Integrins and associated focal adhesion (FA) proteins form a tension-sensitive mechanical link between the extracellular matrix (ECM) and the cytoskeleton, and serve as key components in the signaling cascade by which cells transduce mechanical signals into biological responses (mechanotransduction) (1, 2). Contractile stresses generated by the cell are balanced by tractions at cell–substrate adhesions, and the FA protein vinculin accumulates at regions of high substrate stress (3, 4). The FA protein paxillin colocalizes with vinculin (4) and mediates β1-integrin FA turnover through interaction with FA kinase (FAK) (5). The FAK–paxillin signaling axis recruits vinculin to β1 integrins at regions of high matrix adhesion tension (6), and paxillin—a key mechanosensor (7)—mediates protrusion formation at regions of high stress on 2D substrates (8), and FAK–paxillin–vinculin signaling is required for mechanosensing and durotaxis (9).The tumor microenvironment imparts mechanical and chemical signals on tumor and stromal cells (10), and advanced breast carcinomas are characterized by high interstitial fluid pressure (11), an indicator of poor prognosis (12). This elevated fluid pressure drives interstitial flow (IF) and alters chemical transport within the tumor (13), and IF influences tumor cell migration through the generation of autocrine chemokine gradients (14). Equally important, although not as well understood, is the physical drag imparted on the ECM and constitutive cells (15) by IF, which is analogous to the FA-activating shear stresses generated on endothelial cells by hemodynamic forces (16). With endothelial cells, shear stress can be the dominant mechanical stimulus that induces FAK activation and cytoskeletal remodeling; however, for cells embedded within a porous matrix scaffold, the ratio of the force due to the pressure drop across the cell to the total shear force is inversely proportional to hydrogel permeability (SI Appendix, Eq. S5). In this study, we recapitulate physiologically relevant IF through collagen gel within a microfluidic device. Because the permeability of the collagen I hydrogel used in this study is small (1 × 10⁻¹³ m²), the integrated pressure force is more than 30× the integrated shear force for a 20-μm-diameter cell (17) (SI Appendix, Eq. S5). To maintain static equilibrium, all fluid stresses imparted on the cell must be balanced by tension in matrix adhesions. In 2D, the adhesions balancing the fluid drag on the cell are confined to the basal cell surface, whereas in porous media, such as breast stromal ECM, matrix adhesions are distributed across the full cell surface. Consequently, maintaining static equilibrium requires greater adhesion tension on the upstream side of the cell to balance fluid stresses. From the reference frame of the cell, the effect of IF is mechanically equivalent to applying a net outward force at matrix adhesions on the upstream side of the cell, similar to the net tensile stresses applied by use of optical tweezers to study the molecular mechanisms underlying mechanotransduction (4, 18).Here, we demonstrate that the forces required to balance drag imparted on the cell by IF induce a transcellular gradient in matrix adhesion tension, and the tensile stresses at the upstream side of the cell induce FA reorganization and polarization of FA-plaque proteins including vinculin, paxillin, FAK, FAK^PY397, and α-actinin. FA polarization leads to paxillin-dependent actin localization, the formation of protrusions upstream, and rheotaxis. Consistent with the governing mechanism of durotaxis on 2D substrates, this 3D mechanotransduction occurs through FAK and requires paxillin. Importantly, silencing paxillin does not affect cell migration speed but does attenuate rheotaxis. IF is present in many tissues in vivo (19), and because FA polarization and rheotaxis result from a mechanical force balance, this 3D mechanotransduction mechanism may be fundamental to all cells embedded within porous ECM.     

Structural brain changes in prenatal methamphetamine-exposed children

The global use of methamphetamine (MA) has increased substantially in recent years, but the effect of MA on brain structure in prenatally exposed children is understudied. Here we aimed to investigate potential changes in brain volumes and cortical thickness of children with prenatal MA-exposure compared to unexposed controls. Eighteen 6-year old children with MA-exposure during pregnancy and 18 healthy controls matched for age, gender and socio-economic background underwent structural imaging. Brain volumes and cortical thickness were assessed using Freesurfer and compared using ANOVA. Left putamen volume was significantly increased, and reduced cortical thickness was observed in the left hemisphere of the inferior parietal, parsopercularis and precuneus areas of MA-exposed children compared to controls. Compared to control males, prenatal MA-exposed males had greater volumes in striatal and associated areas, whereas MA-exposed females predominantly had greater cortical thickness compared to control females. In utero exposure to MA results in changes in the striatum of the developing child. In addition, changes within the striatal, frontal, and parietal areas are in part gender dependent.