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Background: Pathologic tissue remodeling with scarring and tissue rigidity has been demonstrated in inflammatory, autoimmune, and allergic diseases. Eosinophilic esophagitis (EoE) is an allergic disease that is diagnosed and managed by repeated biopsy procurement, allowing an understanding of tissue fibroblast dysfunction. While EoE-associated tissue remodeling causes clinical dysphagia, food impactions, esophageal rigidity, and strictures, molecular mechanisms driving these complications remain under investigation., Objective: We hypothesized that chronic EoE inflammation induces pathogenic fibroblasts with dysfunctional tissue regeneration and motility., Methods: We used single-cell RNA sequencing, fluorescence-activated cell sorting analysis, and fibroblast differentiation and migration assays to decipher the induced and retained pathogenic dysfunctions in EoE versus healthy esophageal fibroblasts., Results: Differentiation assays demonstrated that active EoE fibroblasts retain regenerative programs for rigid cells such as chondrocytes (P < .05) but lose healthy fibroblast capacity for soft cells such as adipocytes (P < .01), which was reflected in biopsy sample immunostaining (P < .01). EoE, but not healthy, fibroblasts show proinflammatory and prorigidity transcriptional programs on single-cell RNA sequencing. In vivo, regenerative fibroblasts reside in perivascular regions and near the epithelial junction, and during EoE, they have significantly increased migration (P < .01). Flow analysis and functional assays demonstrated that regenerative EoE fibroblasts have decreased surface CD73 expression and activity (both P < .05) compared to healthy controls, indicating aberrant adenosine triphosphate handling. EoE fibroblast dysfunctions were induced in healthy fibroblasts by reducing CD73 activity and rescued in EoE using adenosine repletion., Conclusion: A normalization of perturbed extracellular adenosine triphosphate handling and CD73 could improve pathogenic fibroblast dysfunction and tissue regeneration in type 2 inflammatory diseases., Competing Interests: Disclosure statement Supported by grants from the National Institutes of Health (NIH), the National Institute of Allergy and Infectious Diseases R01AI092135, R56AI092135, K24AI135034 (all to S.S.A.), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) R01DK114457 (S.S.A., M.C.), the Campaign Urging Research for Eosinophilic Disease Foundation (S.S.A. and J.B.W.), and the MRD Endowed Chair, Department of Pediatrics, UCSD (S.S.A.). Partially supported by NIH grant UL1TR001442 (UCSD CTSA), NIDDK grant P30 DK120515 (UCSD San Diego Digestive Disease Research Center), the Novo Nordisk Foundation (Center for Biosustainability, Technical University of Denmark) NNF10CC1016517 (A.W.T.C.), R35 GM119850 (N.E.L.), R35GM128938 (F.A.), Ann & Robert H. Lurie Children’s Hospital of Chicago (J.B.W.), NIH U19AI166059 (B.C.), KL2TR002552 (E.M.A.), and K12TR004410 (E.M.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Disclosure of potential conflict of interest: E. M. Abud serves on the scientific advisory boards of Neucyte and Stem Pharm; and is a coinventor of patent WO/2018/160496 (microglia differentiation). S. S. Aceves and R. Dohil are coinventors of budesonide oral suspension (Eohilia), patented by the University of California, San Diego, and licensed and sold by Takeda. S. S. Aceves has consulting agreements with Regeneron-Sanofi and Ferring Pharmaceuticals; and has received funding from Sanofi, Implicit Biosciences, and Bristol Meyers Squibb. B. Croker and N. E. Lewis have received funding from Sanofi. J. L. Kennedy has consulting agreements with Genentech. J. B. Wechsler has consulting agreements with AstraZeneca, Regeneron-Sanofi, and InVenea Therapeutics. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Multipotent cells derived from white adipose tissue have been shown to differentiate into multiple lineages including neurogenic lineages. However, the high innervation of brown adipose tissue by the sympathetic nervous system suggest it might be a better source of neural precursor cells. To investigate potential differences between white and brown progenitors, we cultured white and brown dedifferentiated fat (wDFAT and brDFAT) cells from mouse and human adipose tissue and compared marker expression of neural precursors, and neuronal and glial cells, using fluorescence-activated cell sorting, bright-field imaging, immunofluorescence, and RNA analysis by qPCR. The results showed that both wDFAT and brDFAT cells had the capacity to generate neuronal and glial-like cells under neurogenic conditions. However, the brDFAT cells exhibited enhanced propensity for neurogenic differentiation. The neurogenic cells were at least in part derived from Adiponectin-expressing cells. TdTomato-expressing cells derived from Adiponectin (Adipoq) Cre ERT2 -tdTomato flox/flox mice gave rise to individual cells and cell clusters with neurogenic characteristics. Moreover, human brDFAT cells demonstrated a similar ability to undergo neurogenic differentiation after treatment with neurogenic medium, as assessed by immunofluorescence and qPCR. Together, our results support that brDFAT cells have ability to undergo neurogenic differentiation., (© 2022. The Author(s).)

Rationale: The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations., Objective: A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth., Methods and Results: Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing., Conclusions: Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.

Objective: Obesity has increased to pandemic levels and enhanced understanding of adipose regulation is required for new treatment strategies. Although bone morphogenetic proteins (BMPs) influence adipogenesis, the effect of BMP antagonists such as Noggin is largely unknown. The aim of the study was to define the role of Noggin, an extracellular BMP inhibitor, in adipogenesis., Methods: We generated adipose-derived progenitor cells and a mouse model with adipocyte-specific Noggin deletion using the Adiponectin Cre transgenic mouse, and determined the adipose phenotype of Noggin-deficiency., Results: Our studies showed that Noggin is expressed in progenitor cells but declines in adipocytes, possibly allowing for lipid accumulation. Correspondingly, adipocyte-specific Noggin deletion in vivo promoted age-related obesity in both genders with no change in food intake. Although the loss of Noggin caused white adipose tissue hypertrophy, and whitening and impaired function in brown adipose tissue in both genders, there were clear gender differences with the females being most affected. The females had suppressed expression of brown adipose markers and thermogenic genes including peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1alpha) and uncoupling protein 1 (UCP1) as well as genes associated with adipogenesis and lipid metabolism. The males, on the other hand, had early changes in a few BAT markers and thermogenic genes, but the main changes were in the genes associated with adipogenesis and lipid metabolism. Further characterization revealed that both genders had reductions in VO 2 , VCO 2, and RER, whereas females also had reduced heat production. Noggin was also reduced in diet-induced obesity in inbred mice consistent with the obesity phenotype of the Noggin-deficient mice., Conclusions: BMP signaling regulates female and male adipogenesis through different metabolic pathways. Modulation of adipose tissue metabolism by select BMP antagonists may be a strategy for long-term regulation of age-related weight gain and obesity., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The bone morphogenetic proteins (BMPs) belong to the same superfamily as related to transforming growth factor β (TGFβ), growth and differentiation factors (GDFs), and activins. They were initially described as inducers of bone formation but are now known to be involved in morphogenetic activities and cell differentiation throughout the body, including the development of adipose tissue and adipogenic differentiation. BMP4 and BMP7 are the most studied BMPs in adipose tissue, with major roles in white adipogenesis and brown adipogenesis, respectively, but other BMPs such as BMP2, BMP6, and BMP8b as well as some inhibitors and modulators have been shown to also affect adipogenesis. It has become ever more important to understand adipose regulation, including the BMP pathways, in light of the strong links between obesity and metabolic and cardiovascular disease. In this review, we summarize the available information on BMP signaling in adipose tissue using preferentially articles that have appeared in the last decade, which together demonstrate the importance of BMP signaling in adipose biology., (© 2019 World Obesity Federation.)

Multipotent cells derived from white mature adipocytes, referred to as dedifferentiated fat (DFAT) cells have the capacity differentiate into endothelial cells. The objective of this study was to modify the isolation method for DFAT cells in order to optimize the endothelial lineage potential. The adipocytes were preincubated for 24 hours, washed, and then incubated for 5 days to allow the generated DFAT cells to remain in proximity to the adipocytes while the cells aggregated into cell clusters. The DFAT cells rapidly differentiated into adipocytes after which endothelial-like cells (ECs) emerged and formed tube-like structure closely associated with the newly differentiated adipocytes. The lipid-filled cells then gradually disappeared whereas the network of tube structure expanded over the course of 3 weeks. ECs accounted for 35-45% of the cells derived from the DFAT cells, as assessed by qPCR, immunofluorescence and fluorescence-activated cell sorting. The DFAT cell-derived ECs could also be further enriched by magnetic sorting, thereby serving as a mouse cell line for further research.

Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 (CV2, also known as BMP endothelial cell precursor derived regulator [BMPER]) is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells., (© 2017 Wiley Periodicals, Inc.)

The vascular endothelium is critical for induction of appropriate lineage differentiation in organogenesis. In this study, we report that dysfunctional pulmonary endothelium, resulting from the loss of matrix Gla protein (MGP), causes ectopic hepatic differentiation in the pulmonary epithelium. We demonstrate uncontrolled induction of the hepatic growth factor (HGF) caused by dysregulated cross talk between pulmonary endothelium and epithelium in Mgp -null lungs. Elevated HGF induced hepatocyte nuclear factor 4 α (Hnf4a), which competed with NK2 homeobox 1 (Nkx2.1) for binding to forkhead box A2 (Foxa2) to drive hepatic differentiation in Mgp -null airway progenitor cells. Limiting endothelial HGF reduced Hnf4a, abolished interference of Hnf4a with Foxa2, and reduced hepatic differentiation in Mgp -null lungs. Together, our results suggest that endothelial-epithelial interactions, maintained by MGP, are essential in pulmonary cell differentiation., (© 2017 Yao et al.)

Mesenchymal stem cells (MSCs) have attracted the attention of researchers and clinicians for their ability to differentiate into a number of cell types, participate in tissue regeneration, and repair the damaged tissues by producing various growth factors and cytokines, as well as their unique immunoprivilege in alloreactive hosts. The immunomodulatory functions of exogenous MSCs have been widely investigated in immune-mediated inflammatory diseases and transplantation research. However, a harsh environment at the site of tissue injury/inflammation with insufficient oxygen supply, abundance of reactive oxygen species, and presence of other harmful molecules that damage the adoptively transferred cells collectively lead to low survival and engraftment of the transferred cells. Preconditioning of MSCs ex vivo by hypoxia, inflammatory stimulus, or other factors/conditions prior to their use in therapy is an adaptive strategy that prepares MSCs to survive in the harsh environment and to enhance their regulatory function of the local immune responses. This review focuses on a number of approaches in preconditioning human MSCs with the goal of augmenting their capacity to regulate both innate and adaptive immune responses.

The identification of an ideal cell source for tissue regeneration remains a challenge in the stem cell field. The ability of progeny cells to differentiate into other cell types is important for the processes of tissue reconstruction and tissue engineering and has clinical, biochemical or molecular implications. The adaptation of stem cells from adipose tissue for use in regenerative medicine has created a new role for adipocytes. Mature adipocytes can easily be isolated from adipose cell suspensions and allowed to dedifferentiate into lipid-free multipotent cells, referred to as dedifferentiated fat (DFAT) cells. Compared to other adult stem cells, the DFAT cells have unique advantages in their abundance, ease of isolation and homogeneity. Under proper condition in vitro and in vivo, the DFAT cells have exhibited adipogenic, osteogenic, chondrogenic, cardiomyogenc, angiogenic, myogenic, and neurogenic potentials. In this review, we first discuss the phenomena of dedifferentiation and transdifferentiation of cells, and then dedifferentiation of adipocytes in particular. Understanding the dedifferentiation process itself may contribute to our knowledge of normal growth processes, as well as mechanisms of disease. Second, we highlight new developments in DFAT cell culture and summarize the current understanding of DFAT cell properties. The unique features of DFAT cells are promising for clinical applications such as tissue regeneration.

Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte-derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co-localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes., (© 2015 Wiley Periodicals, Inc.)

Rationale: Endothelial cells have the ability to undergo endothelial-mesenchymal transitions (EndMTs), by which they acquire a mesenchymal phenotype and stem cell-like characteristics. We previously found that EndMTs occurred in the endothelium deficient in matrix γ-carboxyglutamic acid protein enabling endothelial cells to contribute cells to vascular calcification. However, the mechanism responsible for initiating EndMTs is not fully understood., Objective: To determine the role of specific serine proteases and sex determining region Y-box 2 (Sox2) in the initiation of EndMTs., Methods and Results: In this study, we used in vivo and in vitro models of vascular calcification to demonstrate that serine proteases and Sox2 are essential for the initiation of EndMTs in matrix γ-carboxyglutamic acid protein-deficient endothelium. We showed that expression of a group of specific serine proteases was highly induced in endothelial cells at sites of vascular calcification in Mgp null aortas. Treatment with serine protease inhibitors decreased both stem cell marker expression and vascular calcification. In human aortic endothelial cells, this group of serine proteases also induced EndMTs, and the activation of proteases was mediated by Sox2. Knockdown of the serine proteases or Sox2 diminished EndMTs and calcification. Endothelial-specific deletion of Sox2 decreased expression of stem cell markers and aortic calcification in matrix γ-carboxyglutamic acid protein-deficient mice., Conclusions: Our results suggest that Sox2-mediated activation of specific serine proteases is essential for initiating EndMTs, and thus, may provide new therapeutic targets for treating vascular calcification., (© 2015 American Heart Association, Inc.)

Mutations in ABCC6 (ATP-binding cassette, subfamily C, member 6), an orphan transporter expressed in the liver, are the cause of pseudoxanthoma elasticum. Since ABCC6 was reported to affect matrix Gla protein (MGP), an inhibitor of bone morphogenetic proteins (BMPs), we studied BMP signaling and expression in various tissues of mice with and without functional ABCC. Enhanced BMP signaling was found in all examined tissues in the absence of ABCC6. Despite this, the expression of particular BMP proteins varied widely between tissues. Interestingly, the expression of most BMP proteins in the liver moved in the opposite direction to the same BMP proteins in kidneys in response to ABCC6 alterations. Thus, ABCC6 deficiency stimulates BMP signaling by acting on the expression of multiple BMPs.

White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5-7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies.

Cerebral arteriovenous malformations (AVMs) are common vascular malformations, which may result in hemorrhagic strokes and neurological deficits. Bone morphogenetic protein (BMP) and Notch signaling are both involved in the development of cerebral AVMs, but the cross-talk between the two signaling pathways is poorly understood. Here, we show that deficiency of matrix Gla protein (MGP), a BMP inhibitor, causes induction of Notch ligands, dysregulation of endothelial differentiation, and the development of cerebral AVMs in MGP null (Mgp(-/-)) mice. Increased BMP activity due to the lack of MGP induces expression of the activin receptor-like kinase 1, a BMP type I receptor, in cerebrovascular endothelium. Subsequent activation of activin receptor-like kinase 1 enhances expression of Notch ligands Jagged 1 and 2, which increases Notch activity and alters the expression of Ephrin B2 and Ephrin receptor B4, arterial and venous endothelial markers, respectively. Reducing the expression of Jagged 1 and 2 in the Mgp(-/-) mice by crossing them with Jagged 1 or 2 deficient mice reduces Notch activity, normalizes endothelial differentiation, and prevents cerebral AVMs, but not pulmonary or renal AVMs. Our results suggest that Notch signaling mediates and can modulate changes in BMP signaling that lead to cerebral AVMs.

Rationale: Vascular calcification is a regulated process that involves osteoprogenitor cells and frequently complicates common vascular disease, such as atherosclerosis and diabetic vasculopathy. However, it is not clear whether the vascular endothelium has a role in contributing osteoprogenitor cells to the calcific lesions., Objective: To determine whether the vascular endothelium contributes osteoprogenitor cells to vascular calcification., Methods and Results: In this study, we use 2 mouse models of vascular calcification, mice with gene deletion of matrix Gla protein, a bone morphogenetic protein (BMP)-inhibitor, and Ins2Akita/+ mice, a diabetes model. We show that enhanced BMP signaling in both types of mice stimulates the vascular endothelium to contribute osteoprogenitor cells to the vascular calcification. The enhanced BMP signaling results in endothelial-mesenchymal transitions and the emergence of multipotent cells, followed by osteoinduction. Endothelial markers colocalize with multipotent and osteogenic markers in calcified arteries by immunostaining and fluorescence-activated cell sorting. Lineage tracing using Tie2-Gfp transgenic mice supports an endothelial origin of the osteogenic cells. Enhancement of matrix Gla protein expression in Ins2Akita/+ mice, as mediated by an Mgp transgene, limits the generation of multipotent cells. Moreover, matrix Gla protein-depleted human aortic endothelial cells in vitro acquire multipotency rendering the cells susceptible to osteoinduction by BMP and high glucose., Conclusions: Our data suggest that the endothelium is a source of osteoprogenitor cells in vascular calcification that occurs in disorders with high BMP activation, such as deficiency of BMP-inhibitors and diabetes mellitus.

White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced by the activin receptor like-kinase 1 (ALK1) when stimulated by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation involving ALK2, ALK1, BMP-4, and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression, proliferation, and tube formation in ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in an abnormal endothelium with increased endothelial cellularity and expression of lineage markers for mature endothelial cells. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.

The vasculature, an organ that penetrates every other organ, is ideally poised to be the site where pools of stem cells are placed, to be deployed and committed in response to feedback regulation, and to respond to demands for new vascular structures. These pools of multipotent cells are often under the regulation of various members of the transforming growth factor-β superfamily, including the bone morphogenetic proteins and their antagonists. Regulation of stem cell populations affects their recruitment, differentiation, spatial organization, and their coordination with host tissue. Loss and dysregulation of feedback control cause a variety of diseases that involve ectopic tissue formation, including atherosclerotic lesion formation and calcification, diabetic vasculopathies, and arteriovenous malformations., (Copyright © 2012 AlphaMed Press.)

Arteriovenous malformations (AVMs) in organs, such as the lungs, intestine, and brain, are characteristic of hereditary hemorrhagic telangiectasia (HHT), a disease caused by mutations in activin-like kinase receptor 1 (ALK1), which is an essential receptor in angiogenesis, or endoglin. Matrix Gla protein (MGP) is an antagonist of BMPs that is highly expressed in lungs and kidneys and is regulated by ALK1. The objective of this study was to determine the role of MGP in the vasculature of the lungs and kidneys. We found that Mgp gene deletion in mice caused striking AVMs in lungs and kidneys, where overall small organ size contrasted with greatly increased vascularization. Mechanistically, MGP deficiency increased BMP activity in lungs. In cultured lung epithelial cells, BMP-4 induced VEGF expression through induction of ALK1, ALK2, and ALK5. The VEGF secretion induced by BMP-4 in Mgp-/- epithelial cells stimulated proliferation of ECs. However, BMP-4 inhibited proliferation of lung epithelial cells, consistent with the increase in pulmonary vasculature at the expense of lung tissue in the Mgp-null mice. Similarly, BMP signaling and VEGF expression were increased in Mgp-/- mouse kidneys. We therefore conclude that Mgp gene deletion is what we believe to be a previously unidentified cause of AVMs. Because lack of MGP also causes arterial calcification, our findings demonstrate that the same gene defect has drastically different effects on distinct vascular beds.

Introduction: Previous studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes., Research Design and Methods: Human adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs., Results: Co-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes., Conclusions: Cross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes.

Statins exert pleiotropic effects, including antioxidative and cellular protective effects. Endogenous adrenomedullin (AM) induces anti-inflammatory, anti-fibrotic and proangiogenic effects. We examined the effects of simvastatin on cardiac fibrosis and apoptosis in AM heterozygous knockout (AM(+/-)) mice treated with angiotensin (Ang) II and high salt loading. Seven-week-old AM(+/-) mice were infused with Ang II while on a high-salt diet with or without simvastatin for 2 weeks. Hearts were stained by hematoxylin-eosin or Masson's trichrome, and were immunostained with isolectin B(4) and α-smooth muscle actin antibodies. Expression of c-Kit and Sca-1 messenger RNA (mRNA) was evaluated by real-time PCR analysis. Apoptotic cells in hearts were identified by terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL) staining. Hearts from Ang II/salt loading AM(+/-) mice showed marked perivascular fibrosis around coronary arteries. Treatment with simvastatin significantly inhibited the fibrosis around coronary arteries in Ang II/salt-loading AM(+/-) mice. Expression of c-Kit and Sca-1 mRNAs in hearts from Ang II/salt-loading AM(+/-) mice was significantly lower than in hearts from wild-type mice. Treatment with simvastatin significantly increased the suppressed expression of c-Kit and Sca-1 mRNAs. In addition, treatment with simvastatin significantly increased the number of isolectin B(4)-positive capillary arteries in hearts from Ang II/salt-loading AM(+/-) mice. Ang II/high salt significantly increased apoptotic cells in hearts from AM(+/-) mice; this trend was reversed by treatment with simvastatin. Thus, statins have potent cardioprotective effects that may be associated with anti-fibrotic, proangiogenic and anti-apoptotic effects in Ang II/salt-loading AM(+/-) mice., (© 2011 The Japanese Society of Hypertension All rights reserved)

Rationale: Diabetes mellitus is frequently complicated by cardiovascular disease, such as vascular calcification and endothelial dysfunction, which have been associated with bone morphogenetic proteins (BMPs)., Objective: To determine whether hyperglycemia in vitro and diabetes in vivo promote vascular BMP activity and correlate with vascular calcification., Methods and Results: Increased glucose augmented expression of BMP-2 and BMP-4; the BMP inhibitors matrix Gla protein (MGP) and Noggin; activin-like kinase receptor (ALK)1, -2, -3 and -6; the BMP type 2 receptor; and the vascular endothelial growth factor in human aortic endothelial cells (HAECs). Diabetes induced expression of the same factors in the aortic wall of 3 animal models of diabetes, Ins2(Akita/+) mice, db/db mice, and HIP rats (rats transgenic for human islet amyloid polypeptide), representative of types 1 and 2 diabetes. Conditioned media from glucose-treated HAECs increased angiogenesis in bovine aortic endothelial cells, as mediated by BMP-4, and osteogenesis in calcifying vascular cells, as mediated by BMP-2. BMP-4, MGP, ALK1, and ALK2 were predominantly expressed on the endothelial side of the aorta, and small interfering RNA experiments showed that these genes were regulated as a group. Diabetic mice and rats showed a dramatic increase in aortic BMP activity, as demonstrated by SMAD1/5/8 phosphorylation. This was associated with increased osteogenesis and calcium accumulation. These changes were prevented in the Ins2(Akita/+) mice by breeding them with MGP transgenic mice, which increased aortic BMP inhibition., Conclusions: Hyperglycemia and diabetes activate vascular BMP activity, which is instrumental in promoting vascular calcification and may be limited by increasing BMP inhibition.

Background: Although transplantation of mononuclear cells (MNCs) induces angiogenesis in myocardial infarction, transplantation requires a large amount of bone marrow or peripheral blood cells. We examined the effects of transplantation of peripheral MNCs expressing an exogenous vascular endothelial growth factor (VEGF) gene in a pig model of acute myocardial infarction (AMI)., Methods: MNCs were isolated from 20 ml peripheral blood from pigs and transfected with 10 microg of human VEGF165 plasmid (phVEGF). Myocardial infarction was induced by occlusion of the mid portion of the left anterior descending coronary artery (LAD) in anesthetized pigs. At 4 h after total occlusion, 5 x 10(6) VEGF-transfected MNCs were retrogradely transplanted into the pig via the coronary vein. Cardiac function, neovascularization and histology of the ischemic tissue were evaluated 4 weeks after transplantation., Results: MNCs expressing hVEGF and infused via the coronary vein were efficiently delivered the heart in pigs with myocardial infarction. Transplantation of MNCs expressing hVEGF significantly increased left ventricular (LV) function, collateral vessels, and capillary density in heart from AMI model pigs. Transplantation of MNCs expressing hVEGF increased the wall thickness of the scar in the heart after AMI., Conclusions: Retrograde transplantation of peripheral blood MNCs expressing hVEGF efficiently induced angiogenesis and improved the impaired LV function in hearts of pigs with AMI. These findings indicate that angiogenic cells and gene therapy may be useful to treat ischemic heart disease., (Copyright 2008 Elsevier Ireland Ltd. All rights reserved.)

Aims: Adipose stromal cells and dissociated brown adipose tissue have been shown to generate cardiomyocyte-like cells. However, it is not clear whether white mature adipocytes have the same potential, even though a close relationship has been found between adipocytes and vascular endothelial cells, another cardiovascular cell type. The objective of this study was to examine if white adipocytes would be able to supply cardiomyocytes., Methods and Results: We prepared a highly purified population of lipid-filled adipocytes from mice, 6-7 weeks of age. When allowed to lose lipids, the adipocytes assumed a fibroblast-like morphology, so-called dedifferentiated fat (DFAT) cells. Subsequently, 10-15% of the DFAT cells spontaneously differentiated into cardiomyocyte-like cells, in which the cardiomyocyte phenotype was identified by morphological observations, expression of cardiomyocyte-specific markers, and immunocytochemical staining. In addition, electrophysiological studies revealed pacemaker activity in these cells, and functional studies showed that a beta-adrenergic agonist stimulated the beating rate, whereas a beta-antagonist reduced it. In vitro treatment of newly isolated adipocytes or DFAT cells with inhibitors of bone morphogenetic proteins (BMP) and Wnt signalling promoted the development of the cardiomyocyte phenotype as determined by the number or beating colonies of cardiomyocyte-like cells and expression of troponin I, a cardiomyocyte-specific marker. Inhibition of BMP was most effective in promoting the cardiomyocyte phenotype in adipocytes, whereas Wnt-inhibition was most effective in DFAT cells., Conclusion: White mature adipocytes can differentiate into cardiomyocyte-like cells, suggesting a link between adipocyte and cardiomyocyte differentiation.

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.

Objective: High-density lipoproteins (HDL) have antiinflammatory effects on the vascular endothelium. Because bone morphogenetic proteins (BMPs) are known to be inflammatory mediators, we examined the effect of HDL on BMP signaling., Methods and Results: Increasing concentrations of HDL progressively enhanced expression of the activin-like kinase receptor (ALK)1 and ALK2 in human aortic endothelial cells as determined by real-time polymerase chain reaction and immunoblotting. Induction of ALK1 was a result of enhanced ALK2 expression as determined by siRNA interference, and was associated with increased levels of vascular endothelial growth factor (VEGF) and matrix Gla protein (MGP). The HDL-induction of ALK2 was dependent on BMP-signaling, and affected coregulation of the ALK2 gene by the homeodomain proteins MSX2, DLX3, and DLX5, as determined by reporter gene assays, siRNA interference, and chromatin immunoprecipitation. Apolipoprotein A-I transgenic mice, known to have high HDL and inhibition of atherogenesis, exhibited similar changes in aortic gene expression as seen in endothelial cells treated with HDL in vitro., Conclusions: We conclude that HDL benefits the arterial wall by allowing for enhanced ALK1 and ALK2 signaling, resulting in an increase of VEGF and MGP, essential for endothelial cell survival and prevention of vascular calcification, respectively.

The Uygur are reported to have an unusually long life expectancy. The purpose of this research was to perform a time-series analysis of systolic blood pressure (SBP) variations in the Uygur and clarify the role of blood pressure variation (BPV) in their longevity. A cross-sectional survey was carried out in Hotan. We investigated 133 clinically healthy elderly Uygur subjects and divided them into two groups: 1) 33 Uygur centenarians in Hotan (UCH; aged > or = 100 years); and 2) 100 elderly Uygur in Hotan (UEH; aged 65-70 years). Blood pressure (BP) was monitored and analyzed with ambulatory BP monitoring. The frequency domain measures were obtained with the maximum entropy method. The mean 24-h SBP was higher in UCH than in UEH. The ratio of non-dipper type BPV was larger in the UCH than in UEH. The highest power spectral density occurred over a 12-h rather than a 24-h period in both UCH and UEH. Ultradian BPVs were more frequent in UCH than in UEH. The least square-fitting curves demonstrated that the maximum values, minimum values, and mean 24-h SBP values were higher in UCH than in UEH. The higher BP and greater number of ultradian BPVs in UCH may have been due to the greater energy expenditure for maintaining daily activities in this population. Factors such as meals, daytime naps, nocturnal micturition, decreased baroreceptor sensitivity, and arterial sclerosis may also have contributed to the higher ultradian BPVs. In conclusion, BPV in the 12-h is more dominant than in the 24-h in both UCH and UEH. BPVs in the 3-h and 4-h are more frequent in UCH than those in UEH.

Cross-sectional surveillance was carried out in long-lived Uygur in China to investigate blood pressure (BP) and pulse rate (PR) variation in centenarians. The study group comprised 33 centenarians (age > or = 100 years), 103 longevous subjects (age 90-99 years) and 100 elderly (age 65-70 years) subjects. Office BP was measured, and 24-h noninvasive ambulatory blood pressure monitoring (ABPM) was performed. The office BP was higher and hypertension occurred more frequently in the centenarians than in either the longevous or elderly subjects. Mean 24-h systolic and diastolic BP was higher in the centenarians than in the other 2 groups. However, mean 24-h PR did not differ between them. Day-night differences in systolic BP decreased, and the non-dipper-type BP pattern was common in the centenarians (79.1% vs 68% and 63.6% in longevous and elderly subjects, respectively). Circadian BP was characterized by 3 peaks in longevous subjects and multiple peaks in centenarians. Morning rising and nocturnal dipping of BP were observed in both longevous and elderly subjects. In conclusion, BP in longevous and elderly Uygur was characterized by circadian rhythmicity, but the nocturnal fall in BP was not seen in centenarians. Differences in day-night BP and circadian rhythmicity may decrease with advancing age, especially in centenarians.

The Uygur in Hotan (Xinjiang, China) are reported to have a long life expectancy. The purpose of this study was to clarify the relationship between variations in blood pressure (BP) and longevity. Cross-sectional surveillance was carried out in both Hotan and Barkol. The subjects were divided into five groups: 1. Uygur longevity subjects in Hotan (103 subjects, age >90 yr); 2. Uygur elderly subjects in Hotan (107 subjects, age 65-70 yr); 3. Han elderly subjects in Hotan (41 subjects, age 65-70 yr); 4. Kazakh elderly subjects in Barkol (117 subjects, age 65-70 yr); 5. Han elderly subjects in Barkol (50 subjects, age 65-70 yr). BP was monitored and analyzed using the fourteen devices of ambulatory BP monitoring. The prevalence of hypertension was lowest in the Uygur (16.2% in Uygur elderly subjects in Hotan; 23.7% in Uygur longevity subjects in Hotan; 27.0% in Han elderly subjects in Hotan; 42.0% in Han elderly subjects in Barkol; 50.0% in Kazakh elderly subjects in Barkol). The ratio of dips in BP was largest in the Han (57% in Han elderly subjects in Barkol; 50% in Han elderly subjects in Hotan; 50% in Uygur longevity subjects in Hotan, 49% in Uygur elderly subjects in Hotan; 17% in Kazakh elderly subjects in Barkol). The 24-h mean systolic BP in Uygur longevity subjects in Hotan was not different from those in Uygur elderly subjects and Han elderly subjects in Hotan, nor did the 24-h mean diastolic BP differ from those in Uygur elderly subjects and Han elderly subjects in Hotan respectively. In conclusion, Uygur subjects seem to be less hypertensive, compared to Kazakh subjects. Uygur longevity subjects had more dipping in their BP variation than did the Kazakh subjects in Xinjiang, China.