Role of macrophages in regression of myocardial fibrosis following alleviation of left ventricular pressure overload.

Sustained hemodynamic pressure overload (PO) produced by murine transverse aortic constriction (TAC) causes myocardial fibrosis; removal of TAC (unTAC) returns left ventricle (LV) hemodynamic load to normal and results in significant, but incomplete regression of myocardial fibrosis. However, the cellular mechanisms that result in these outcomes have not been defined. The objective was to determine temporal changes in myocardial macrophage phenotype in TAC and unTAC and determine whether macrophage depletion alters collagen degradation after unTAC. Myocardial macrophage abundance and phenotype were assessed by immunohistochemistry, flow cytometry, and gene expression by RT-PCR in control (non-TAC), 2 wk, 4 wk TAC, and 2 wk, 4 wk, and 6 wk unTAC. Myocardial cytokine profiles and collagen-degrading enzymes were determined by immunoassay and immunoblots. Initial collagen degradation was detected with collagen-hybridizing peptide (CHP). At unTAC, macrophages were depleted with clodronate liposomes, and endpoints were measured at 2 wk unTAC. Macrophage number had a defined temporal pattern: increased in 2 wk and 4 wk TAC, followed by increases at 2 wk unTAC (over 4 wk TAC) that then decreased at 4 wk and 6 wk unTAC. At 2 wk unTAC, macrophage area was significantly increased and was regionally associated with CHP reactivity. Cytokine profiles in unTAC reflected a proinflammatory milieu versus the TAC-induced profibrotic milieu. Single-cell sequencing analysis of 2 wk TAC versus 2 and 6 wk unTAC revealed distinct macrophage gene expression profiles at each time point demonstrating unique macrophage populations in unTAC versus TAC myocardium. Clodronate liposome depletion at unTAC reduced CHP reactivity and decreased cathepsin K and proMMP2. We conclude that temporal changes in number and phenotype of macrophages play a critical role in both TAC-induced development and unTAC-mediated partial, but incomplete, regression of myocardial fibrosis.NEW & NOTEWORTHY Our novel findings highlight the dynamic changes in myocardial macrophage populations that occur in response to PO and after alleviation of PO. Our data demonstrated, for the first time, a potential benefit of macrophages in contributing to collagen degradation and the partial regression of interstitial fibrosis following normalization of hemodynamic load.

Patient-Reported Outcomes Improve with a Virtual Diabetes Care Model that Includes Continuous Glucose Monitoring.

Background: The objective was to examine patient-reported outcomes (PROs) associated with access to a virtual clinic model for diabetes care. Methods: Adults with diabetes (N = 234) received virtual care, including support for continuous glucose monitoring (CGM) over a 6-month study period. Care was led by a Certified Diabetes Care and Education Specialist and focused on optimizing self-management skills and response to glucose values observed on CGM. After 6 months of CGM use and access to diabetes education, participants could opt in to another 6 months of follow-up with access to the virtual care team. Participants completed PRO surveys and had health and glycemic measures collected at baseline, 3, 6, and 12 months. Results: Participants with type 1 diabetes (N = 160) were 44 ± 14 years and had mean baseline HbA1c of 61 mmol/mol (7.7%). Participants with type 2 diabetes (N = 74) were 52 ± 12 years and had mean baseline HbA1c of 66 mmol/mol (8.2%). Compared with baseline levels, at 6 months participants experienced less depression, diabetes distress, and hypoglycemic fears while also experiencing greater satisfaction with glucose monitoring, diabetes technology and specifically with CGM, and confidence for managing hypoglycemic (p < 0.05). For participants with type 1 diabetes, more time in the target range for glucose levels (70-180 mg/dL) was associated with less depression, diabetes distress, and hypoglycemic fears. Conclusions: PROs improved for adults with diabetes utilizing virtual diabetes care, including support for CGM use. Paired with the glycemic improvements observed in this virtual clinic study, there were robust benefits on the quality of life of adults with diabetes. ClinicalTrials.gov Identifier: NCT04765358.

Mechanisms that limit regression of myocardial fibrosis following removal of left ventricular pressure overload.

Left ventricular pressure overload (LVPO) can develop from antecedent diseases such as aortic valve stenosis and systemic hypertension and is characterized by accumulation of myocardial extracellular matrix (ECM). Evidence from patient and animal models supports limited reductions in ECM following alleviation of PO, however, mechanisms that control the extent and timing of ECM regression are undefined. LVPO, induced by 4 wk of transverse aortic constriction (TAC) in mice, was alleviated by removal of the band (unTAC). Cardiomyocyte cross-sectional area, collagen volume fraction (CVF), myocardial stiffness, and collagen degradation were assessed for: control, 2-wk TAC, 4-wk TAC, 4-wk TAC + 2-wk unTAC, 4-wk TAC + 4-wk unTAC, and 4-wk TAC + 6-wk unTAC. When compared with 4-wk TAC, 2-wk unTAC resulted in increased reactivity of collagen hybridizing peptide (CHP) (representing initiation of collagen degradation), increased levels of collagenases and gelatinases, decreased levels of collagen cross-linking enzymes, but no change in CVF. When compared with 2-wk unTAC, 4-wk unTAC demonstrated decreased CVF, which did not decline to control values. At 4-wk and 6-wk unTAC, CHP reactivity and mediators of ECM degradation were reduced versus 2-wk unTAC, whereas levels of tissue inhibitor of metalloproteinase (TIMP)-1 increased. ECM homeostasis changed in a time-dependent manner after removal of LVPO and is characterized by early increases in collagen degradation, followed by a later dampening of this process. Tempered ECM degradation with time is predicted to contribute to the finding that normalization of hemodynamic overload alone does not completely regress myocardial fibrosis.NEW & NOTEWORTHY In this study, a murine model demonstrated persistent interstitial fibrosis and myocardial stiffness following alleviation of pressure overload.

Ensemble machine learning model identifies patients with HFpEF from matrix-related plasma biomarkers.

Arterial hypertension can lead to structural changes within the heart including left ventricular hypertrophy (LVH) and eventually heart failure with preserved ejection fraction (HFpEF). The initial diagnosis of HFpEF is costly and generally based on later stage remodeling; thus, improved predictive diagnostic tools offer potential clinical benefit. Recent work has shown predictive value of multibiomarker plasma panels for the classification of patients with LVH and HFpEF. We hypothesized that machine learning algorithms could substantially improve the predictive value of circulating plasma biomarkers by leveraging more sophisticated statistical approaches. In this work, we developed an ensemble classification algorithm for the diagnosis of HFpEF within a population of 480 individuals including patients with HFpEF, patients with LVH, and referent control patients. Algorithms showed strong diagnostic performance with receiver-operating-characteristic curve (ROC) areas of 0.92 for identifying patients with LVH and 0.90 for identifying patients with HFpEF using demographic information, plasma biomarkers related to extracellular matrix remodeling, and echocardiogram data. More impressively, the ensemble algorithm produced an ROC area of 0.88 for HFpEF diagnosis using only demographic and plasma panel data. Our findings demonstrate that machine learning-based classification algorithms show promise as a noninvasive diagnostic tool for HFpEF, while also suggesting priority biomarkers for future mechanistic studies to elucidate more specific regulatory roles.NEW & NOTEWORTHY Machine learning algorithms correctly classified patients with heart failure with preserved ejection fraction with over 90% area under receiver-operating-characteristic curves. Classifications using multidomain features (demographics and circulating biomarkers and echo-based ventricle metrics) proved more accurate than previous studies using single-domain features alone. Excitingly, HFpEF diagnoses were generally accurate even without echo-based measurements, demonstrating that such algorithms could provide an early screening tool using blood-based measurements before sophisticated imaging.

Lysyl oxidase directly contributes to extracellular matrix production and fibrosis in systemic sclerosis.

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Lysyl oxidase (LOX) is a copper-dependent amine oxidase whose primary function is the covalent crosslinking of collagens in the extracellular matrix (ECM). We investigated the role of LOX in the pathophysiology of SSc. LOX mRNA and protein levels were increased in lung fibroblasts of SSc patients compared with healthy controls and IPF patients. In vivo, bleomycin induced LOX mRNA expression in lung tissues, and LOX activity increased in the circulation of mice with pulmonary fibrosis, suggesting that circulating LOX parallels levels in lung tissues. Circulating levels of LOX were reduced upon amelioration of fibrosis with an antifibrotic peptide. LOX induced ECM production at the transcriptional level in lung fibroblasts, human lungs, and human skin maintained in organ culture. In vivo, LOX synergistically exacerbated fibrosis in bleomycin-treated mice. Further, LOX increased the production of interleukin (IL)-6, and the increase was mediated by LOX-induced c-Fos expression, the nuclear localization of c-Fos, and its engagement with the IL-6 promoter region. Our findings demonstrate that LOX expression and activity correlate with fibrosis in vitro, ex vivo, and in vivo. LOX induced ECM production via upregulation of IL-6 and nuclear localization of c-Fos. Thus, LOX has a direct pathogenic role in SSc-associated fibrosis that is independent of its crosslinking function. Our findings also suggest that measuring circulating LOX levels and activity can be used for monitoring response to antifibrotic therapy.

SPARC production by bone marrow-derived cells contributes to myocardial fibrosis in pressure overload.

In human heart failure and in murine hearts with left-ventricular pressure overload (LVPO), increases in fibrosis are associated with increases in myocardial stiffness. Secreted protein acidic and rich in cysteine (SPARC) is shown to be necessary for both cardiac fibrosis and increases in myocardial stiffness in response to LVPO; however, cellular sources of cardiac SPARC are incompletely defined. Irradiation and bone marrow transfer were undertaken to test the hypothesis that SPARC expression by bone marrow-derived cells is an important mediator of fibrosis in LVPO. In recipient SPARC-null mice transplanted with donor wild-type (WT) bone marrow and subjected to LVPO, levels of fibrosis similar to that of WT mice were found despite the lack of SPARC expression by resident cells. In recipient WT mice with donor SPARC-null bone marrow, significantly less fibrosis versus that of WT mice was found despite the expression of SPARC by resident cells. Increases in myocardial stiffness followed a similar pattern to that of collagen deposition. Myocardial macrophages were significantly reduced in SPARC-null mice with LVPO versus that of WT mice. Recipient SPARC-null mice transplanted with donor WT bone marrow exhibited an increase in cardiac macrophages versus that of SPARC-null LVPO and donor WT mice with recipient SPARC-null bone marrow. Expression of vascular cellular adhesion molecule (VCAM), a previously identified binding partner of SPARC, was assessed in all groups and with the exception of WT mice, increases in VCAM immunoreactivity with LVPO were observed. However, no differences in VCAM expression between bone marrow transplant groups were noted. In conclusion, SPARC expression by bone marrow-derived cells was critical for fibrotic deposition of collagen and influenced the expansion of myocardial macrophages in response to LVPO.NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in LV and myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, a murine model of cardiac fibrosis induced by PO was used to demonstrate a critical function of SPARC in bone marrow-derived cells that drives cardiac fibrosis and increases in cardiac macrophages.

Pressure overload generates a cardiac-specific profile of inflammatory mediators.

Mechanisms that contribute to myocardial fibrosis, particularly in response to left ventricular pressure overload (LVPO), remain poorly defined. To test the hypothesis that a myocardial-specific profile of secreted factors is produced in response to PO, levels of 44 factors implicated in immune cell recruitment and function were assessed in a murine model of cardiac hypertrophy and compared with levels produced in a model of pulmonary fibrosis (PF). Mice subjected to PO were assessed at 1 and 4 wk. Protein from plasma, LV, lungs, and kidneys were analyzed by specific protein array analysis in parallel with protein from mice subjected to silica-instilled PF. Of the 44 factors assessed, 13 proteins were elevated in 1-wk PO myocardium, whereas 18 proteins were found increased in fibrotic lung. Eight of those increased in 1-wk LVPO were not found to be increased in fibrotic lungs (CCL-11, CCL-12, CCL-17, CCL-19, CCL-21, CCL-22, IL-16, and VEGF). Additionally, six factors were increased in plasma of 1-wk LVPO in the absence of increases in myocardial levels. In contrast, in mice with PF, no factors were found increased in plasma that were not elevated in lung tissue. Of those factors increased at 1 wk, only TIMP-1 remained elevated at 4 wk of LVPO. Immunohistochemistry of myocardial vasculature at 1 and 4 wk revealed similar amounts of total vasculature; however, evidence of activated endothelium was observed at 1 wk and, to a lesser extent, at 4 wk LVPO. In conclusion, PO myocardium generated a unique signature of cytokine expression versus that of fibrotic lung.NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, cytokine profiles produced in a murine model of cardiac fibrosis induced by PO were compared with those produced in response to silica-induced lung fibrosis. A unique profile of cardiac tissue-specific and plasma-derived factors generated in response to PO are reported.

Focusing Heart Failure Research on Myocardial Fibrosis to Prioritize Translation.

Heart failure (HF) has traditionally been defined by symptoms of fluid accumulation and poor perfusion, but it is now recognized that specific HF classifications hold prognostic and therapeutic relevance. Specifically, HF with reduced ejection fraction is characterized by reduced left ventricular systolic pump function and dilation and HF with preserved ejection fraction is characterized primarily by abnormal left ventricular filling (diastolic failure) with relatively preserved left ventricular systolic function. These forms of HF are distributed equally among patients with HF and likely require distinctly different strategies to mitigate the morbidity, mortality, and medical resource utilization of this disease. In particular, HF is a significant medical issue within the US Department of Veterans Affairs (VA) hospital system and constitutes a major translational research priority for the VA. Because a common underpinning of both HF with reduced ejection fraction and HF with preserved ejection fraction seems to be changes in the structure and function of the myocardial extracellular matrix, a conference was convened sponsored by the VA, entitled, "Targeting Myocardial Fibrosis in Heart Failure" to explore the extracellular matrix as a potential therapeutic target and to propose specific research directions. The conference was conceptually framed around the hypothesis that although HF with reduced ejection fraction and HF with preserved ejection fraction clearly have distinct mechanisms, they may share modifiable pathways and biological mediators in common. Inflammation and extracellular matrix were identified as major converging themes. A summary of our discussion on unmet challenges and possible solutions to move the field forward, as well as recommendations for future research opportunities, are provided.

Inhibition of transglutaminase activity in periodontitis rescues periodontal ligament collagen content and architecture.

BACKGROUND AND OBJECTIVE: Periodontal disease (PD) afflicts approximately 50% of the population in the United States and is characterized by chronic inflammation of the periodontium that can lead to loss of the periodontal ligament through collagen degradation, loss of alveolar bone, and to eventual tooth loss. Previous studies have implicated transglutaminase (TG) activity in promoting thin collagen I fiber morphology and decreased mechanical strength in homeostatic PDL. The aim of this study was to determine whether TG activity influenced collagen assembly in PDL in the setting of periodontal disease. MATERIAL AND METHODS: A ligature model was used to induce clinically relevant PD in mice. Mice with ligature were assessed at 5 and 14 days to determine PDL collagen morphology, transglutaminase (TG) activity, and bone loss. The effects of inhibition of TG on PDL were assessed by immunohistochemistry and second-harmonic generation (SHG) to visualize collagen fibers in native tissue. RESULTS: Ligature placement around the 2nd molar resulted in significant bone loss and a decrease in total collagen content after 5 days of ligature placement. A significant increase in thin over thick fibers was also demonstrated in mice with ligature at 5 days associated with apparent increases in immunoreactivity for TG2 and for TG-mediated N-ε-γ-glutamyl cross-links in PDL. Inhibition of TG activity increased total collagen and thick collagen fiber content over vehicle control in mice with ligature for 5 days. SHG of PDL was used to visualize and quantify the effects of TG inhibition on enhanced collagen fiber organization in unfixed control and diseased PDL. CONCLUSION: These studies support a role of TG in regulating collagen fiber assembly and suggest that strategies to inhibit TG activity in disease might contribute to restoration of PDL tissue integrity.

Increased macrophage-derived SPARC precedes collagen deposition in myocardial fibrosis.

Myocardial fibrosis and the resultant increases in left ventricular stiffness represent pivotal consequences of chronic pressure overload (PO) that impact both functional capacity and the rates of morbid and mortal events. However, the time course and cellular mechanisms that underlie PO-induced fibrosis have not been completely defined. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that has been shown to be required for insoluble collagen deposition and increased myocardial stiffness in response to PO in mice. As macrophages are associated with increases in fibrillar collagen, the hypothesis that macrophages represent a source of increased SPARC production in the PO myocardium was tested. The time course of changes in the myocardial macrophage population was compared with changes in procollagen type I mRNA, production of SPARC, fibrillar collagen accumulation, and diastolic stiffness. In PO hearts, mRNA encoding collagen type I was increased at 3 days, whereas increases in levels of total collagen protein did not occur until 1 wk and were followed by increases in insoluble collagen at 2 wk. Increases in muscle stiffness were not detected before increases in insoluble collagen content (>1 wk). Significant increases in myocardial macrophages that coincided with increased SPARC were found but did not coincide with increases in mRNA encoding collagen type I. Furthermore, immunohistochemistry and flow cytometry identified macrophages as a cellular source of SPARC. We conclude that myocardial macrophages play an important role in the time-dependent increases in SPARC that enhance postsynthetic collagen processing, insoluble collagen content, and myocardial stiffness and contribute to the development of fibrosis. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in left ventricular and myocardial stiffness represent pivotal consequences of chronic pressure overload. In this study a murine model of cardiac fibrosis induced by pressure overload was used to establish a time course of collagen expression, collagen deposition, and cardiac macrophage expansion.

The role of secreted protein acidic and rich in cysteine (SPARC) in cardiac repair and fibrosis: Does expression of SPARC by macrophages influence outcomes?

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular, collagen-binding protein. Matricellular proteins are described as extracellular matrix-associated proteins that do not serve classical structural roles in the matrix such as those ascribed to laminins and collagens. The family of matricellular proteins modulates cell:extracellular matrix interactions and is actively expressed during tissue remodeling. Functional activities attributed to SPARC in cultured cells include regulation of cell adhesion, cytoskeletal rearrangement, proliferation, and matrix assembly. The primary phenotype characteristic of SPARC-null mice is a deficit in amounts of fibrillar collagen and fibril morphology. Strikingly, SPARC-null mice demonstrate a blunted fibrotic response in a number of different tissue settings. The role of monocyte/macrophages as an important component of tissue fibrosis is becoming increasingly appreciated. Expression of SPARC by bone marrow derived inflammatory cells raises the interesting proposition that SPARC produced by infiltrating leukocytes might contribute to the course of inflammation and tissue fibrosis in the heart. This review will summarize results from studies defining the function of SPARC in myocardial repair and fibrosis and results from other non-cardiac tissues that shed light onto possible consequences of SPARC expression by monocyte/macrophages in the setting of heart disease.

Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin.

BACKGROUND: The purpose of this study was to determine whether patients with heart failure and a preserved ejection fraction (HFpEF) have an increase in passive myocardial stiffness and the extent to which discovered changes depend on changes in extracellular matrix fibrillar collagen and cardiomyocyte titin. METHODS AND RESULTS: Seventy patients undergoing coronary artery bypass grafting underwent an echocardiogram, plasma biomarker determination, and intraoperative left ventricular epicardial anterior wall biopsy. Patients were divided into 3 groups: referent control (n=17, no hypertension or diabetes mellitus), hypertension (HTN) without (-) HFpEF (n=31), and HTN with (+) HFpEF (n=22). One or more of the following studies were performed on the biopsies: passive stiffness measurements to determine total, collagen-dependent and titin-dependent stiffness (differential extraction assay), collagen assays (biochemistry or histology), or titin isoform and phosphorylation assays. In comparison with controls, patients with HTN(-)HFpEF had no change in left ventricular end-diastolic pressure, myocardial passive stiffness, collagen, or titin phosphorylation but had an increase in biomarkers of inflammation (C-reactive protein, soluble ST2, tissue inhibitor of metalloproteinase 1). In comparison with both control and HTN(-)HFpEF, patients with HTN(+)HFpEF had increased left ventricular end-diastolic pressure, left atrial volume, N-terminal propeptide of brain natriuretic peptide, total, collagen-dependent, and titin-dependent stiffness, insoluble collagen, increased titin phosphorylation on PEVK S11878(S26), reduced phosphorylation on N2B S4185(S469), and increased biomarkers of inflammation. CONCLUSIONS: Hypertension in the absence of HFpEF did not alter passive myocardial stiffness. Patients with HTN(+)HFpEF had a significant increase in passive myocardial stiffness; collagen-dependent and titin-dependent stiffness were increased. These data suggest that the development of HFpEF depends on changes in both collagen and titin homeostasis.

Increased ADAMTS1 mediates SPARC-dependent collagen deposition in the aging myocardium.

Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein highly expressed during fibrosis. Fibrosis is a prominent component of cardiac aging that reduces myocardial elasticity. Previously, we reported that SPARC deletion attenuated myocardial stiffness and collagen deposition in aged mice. To investigate the mechanisms by which SPARC promotes age-related cardiac fibrosis, we evaluated six groups of mice (n = 5-6/group): young (3-5 mo old), middle-aged (10-12 mo old), and old (18-29 mo old) C57BL/6 wild type (WT) and SPARC-null (Null) mice. Collagen content, determined by picrosirius red staining, increased in an age-dependent manner in WT but not in Null mice. A disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) increased in middle-aged and old WT compared with young, whereas in Null mice only old animals showed increased ADAMTS1 expression. Versican, a substrate of ADAMTS1, decreased with age only in WT. To assess the mechanisms of SPARC-induced collagen deposition, we stimulated cardiac fibroblasts with SPARC. SPARC treatment increased secretion of collagen I and ADAMTS1 (both the 110-kDa latent and 87-kDa active forms) into the conditioned media as well as the cellular expression of transforming growth factor-ß1-induced protein (Tgfbi) and phosphorylated Smad2. An ADAMTS1 blocking antibody suppressed the SPARC-induced collagen I secretion, indicating that SPARC promoted collagen production directly through ADAMTS1 interaction. In conclusion, ADAMTS1 is an important mediator of SPARC-regulated cardiac aging.

Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly.

The ability of the heart to adapt to increased stress is dependent on the modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest that LUM deficient mice (lum(-/-)) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum(-/-) hearts have not been evaluated. These studies show that LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum(-/-) neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum(-/-) hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum(-/-) hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum(-/-) hearts. There was also a reduction in the ß and γ forms of collagenα1(I) in lum(-/-) hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum(-/-) hearts, indicating that LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart. Further investigation into the role of LUM may yield novel therapeutic targets and/or biomarkers for patients with cardiovascular disease.

Secreted protein acidic and rich in cysteine facilitates age-related cardiac inflammation and macrophage M1 polarization.

To investigate the role of secreted protein acidic and rich in cysteine (SPARC) in age-related cardiac inflammation, we studied six groups of mice: young (3-5 mo old), middle-aged (10-12 mo old), and old (18-29 mo old) C57BL/6 wild-type (WT) and SPARC-null (Null) mice (n = 7-10/group). Cardiac function and structure were determined by echocardiography. The left ventricle was used for cytokine gene array and macrophage quantification by immunohistochemistry. Macrophage infiltration increased with age in WT (n = 5-6/group, P < 0.05 for young vs. old), but not in Null. Proinflammatory markers (Ccl5, Cx3cl1, Ccr2, and Cxcr3) increased in middle-aged and old WT, whereas they were increased only in old Null compared with respective young (n = 5-6/group, P < 0.05 for all). These results suggest that SPARC deletion delayed age-related cardiac inflammation. To further assess how SPARC affects inflammation, we stimulated peritoneal macrophages with SPARC (n = 4). SPARC treatment increased expression of proinflammatory macrophage M1 markers and decreased anti-inflammatory M2 markers. Echocardiography (n = 7-10/group) revealed an age-related increase in wall thickness of the left ventricle in WT (0.76 ± 0.02 mm in young vs. 0.91 ± 0.03 mm in old; P < 0.05) but not in Null (0.78 ± 0.01 mm in young vs. 0.84 ± 0.02 mm in old). In conclusion, SPARC deletion delayed age-related increases in macrophage infiltration and proinflammatory cytokine expression in vivo and in vitro. SPARC acts as an important mediator of age-related cardiac inflammation by increasing the expression of macrophage M1 markers and decreasing M2 markers.

Myocardial fibroblast-matrix interactions and potential therapeutic targets.

The cardiac extracellular matrix (ECM) is a dynamic structure, adapting to physiological and pathological stresses placed on the myocardium. Deposition and organization of the matrix fall under the purview of cardiac fibroblasts. While often overlooked compared to myocytes, fibroblasts play a critical role in maintaining ECM homeostasis under normal conditions and in response to pathological stimuli assume an activated, myofibroblast phenotype associated with excessive collagen accumulation contributing to impaired cardiac function. Complete appreciation of fibroblast function is hampered by the lack of fibroblast-specific reagents and the heterogeneity of fibroblast precursors. This is further complicated by our ability to dissect the role of myofibroblasts versus fibroblasts in myocardial in remodeling. This review highlights critical points in the regulation of collagen deposition by fibroblasts, the current panel of molecular tools used to identify fibroblasts and the role of fibroblast-matrix interactions in fibroblast function and differentiation into the myofibroblast phenotype. The clinical potential of exploiting differences between fibroblasts and myofibroblasts and using them to target specific fibroblast populations is also discussed. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium."

IGF-II regulates lysyl oxidase propeptide and mediates its effects in part via basic helix-loop-helix E40.

Pulmonary fibrosis (PF) is a clinically severe and commonly fatal complication of Systemic Sclerosis (SSc). Our group has previously reported profibrotic roles for Insulin-like Growth Factor II (IGF-II) and Lysyl Oxidase (LOX) in SSc-PF. We sought to identify downstream regulatory mediators of IGF-II. In the present work, we show that SSc lung tissues have higher baseline levels of the total (N-glycosylated/unglycosylated) LOX-Propeptide (LOX-PP) than control lung tissues. LOX-PP-mediated changes were consistent with the extracellular matrix (ECM) deregulation implicated in SSc-PF progression. Furthermore, Tolloid-like 1 (TLL1) and Bone Morphogenetic Protein 1 (BMP1), enzymes that can cleave ProLOX to release LOX-PP, were increased in SSc lung fibrosis and the bleomycin (BLM)-induced murine lung fibrosis model, respectively. In addition, IGF-II regulated the levels of ProLOX, active LOX, LOX-PP, BMP1, and isoforms of TLL1. The Class E Basic Helix-Loop-Helix protein 40 (BHLHE40) transcription factor localized to the nucleus in response to IGF-II. BHLHE40 silencing downregulated TLL1 isoforms and LOX-PP, and restored features of ECM deregulation triggered by IGF-II. Our findings indicate that IGF-II, BHLHE40, and LOX-PP may serve as targets of therapeutic intervention to halt SSc-PF progression.

Decellularized heart extracellular matrix alleviates activation of hiPSC-derived cardiac fibroblasts.

Human induced pluripotent stem cell derived cardiac fibroblasts (hiPSC-CFs) play a critical role in modeling human cardiovascular diseases in vitro. However, current culture substrates used for hiPSC-CF differentiation and expansion, such as Matrigel and tissue culture plastic (TCPs), are tissue mismatched and may provide pathogenic cues. Here, we report that hiPSC-CFs differentiated on Matrigel and expanded on tissue culture plastic (M-TCP-iCFs) exhibit transcriptomic hallmarks of activated fibroblasts limiting their translational potential. To alleviate pathogenic activation of hiPSC-CFs, we utilized decellularized extracellular matrix derived from porcine heart extracellular matrix (HEM) to provide a biomimetic substrate for improving hiPSC-CF phenotypes. We show that hiPSC-CFs differentiated and expanded on HEM (HEM-iCFs) exhibited reduced expression of hallmark activated fibroblast markers versus M-TCP-iCFs while retaining their cardiac fibroblast phenotype. HEM-iCFs also maintained a reduction in expression of hallmark genes associated with pathogenic fibroblasts when seeded onto TCPs. Further, HEM-iCFs more homogenously integrated into an hiPSC-derived cardiac organoid model, resulting in improved cardiomyocyte sarcomere development. In conclusion, HEM provides an improved substrate for the differentiation and propagation of hiPSC-CFs for disease modeling.