The ebb and flow of cardiac lymphatics: a tidal wave of new discoveries.

The heart is imbued with a vast lymphatic network that is responsible for fluid homeostasis and immune cell trafficking. Disturbances in the forces that regulate microvascular fluid movement can result in myocardial edema, which has profibrotic and proinflammatory consequences and contributes to cardiovascular dysfunction. This review explores the complex relationship between cardiac lymphatics, myocardial edema, and cardiac disease. It covers the revised paradigm of microvascular forces and fluid movement around the capillary as well as the arsenal of preclinical tools and animal models used to model myocardial edema and cardiac disease. Clinical studies of myocardial edema and their prognostic significance are examined in parallel to the recent elegant animal studies discerning the pathophysiological role and therapeutic potential of cardiac lymphatics in different cardiovascular disease models. This review highlights the outstanding questions of interest to both basic scientists and clinicians regarding the roles of cardiac lymphatics in health and disease.

A 3D macroporous and magnetic Mg2SiO4-CuFe2O4 scaffold for bone tissue regeneration: Surface modification, in vitro and in vivo studies.

Macroporous scaffolds with bioactivity and magnetic properties can be a good candidate for bone regeneration and hyperthermia. In addition, modifying the surface of the scaffolds with biocompatible materials can increase their potential for in vivo applications. Here, we developed a multifunctional nanocomposite Mg2SiO4-CuFe2O4 scaffold for bone regeneration and hyperthermia. The surface of scaffold was coated with various concentrations of poly-3-hydroxybutyrate (P3HB, 1-5% (w/v)). It was observed that 3% (w/v) of P3HB provided a favorable combination of porosity (79 ± 2.1%) and compressive strength (3.2 ± 0.11 MPa). The hyperthermia potential of samples was assessed in the presence of various magnetic fields in vitro. The coated scaffolds showed a lower degradation rate than the un-coated one up to 35 days of soaking in simulated biological medium. Due to the porous and specific morphology of P3HB, it was found that in vitro bioactivity and cell attachment were increased on the scaffold. Moreover, it was observed that the P3HB coating improved the cell viability, alkaline phosphatase activity, and mineralization of the scaffold. Finally, we studied the bone formation ability of the scaffolds in vivo, and implanted the developed scaffold in the rat's femur for 8 weeks. Micro-computed tomography results including bone volume fraction and trabecular thickness exhibited an improvement in the bone regeneration of the coated scaffold compared to the control. The overall results of this study introduce a highly macroporous scaffold with multifunctional performance, noticeable ability in bone regeneration, and hyperthermia properties for osteosarcoma.

VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.

BACKGROUND: The adherens protein VE-cadherin (vascular endothelial cadherin) has diverse roles in organ-specific lymphatic vessels. However, its physiological role in cardiac lymphatics and its interaction with lymphangiogenic factors has not been fully explored. We sought to determine the spatiotemporal functions of VE-cadherin in cardiac lymphatics and mechanistically elucidate how VE-cadherin loss influences prolymphangiogenic signaling pathways, such as adrenomedullin and VEGF (vascular endothelial growth factor)-C/VEGFR3 (vascular endothelial growth factor receptor 3) signaling. METHODS: Cdh5flox/flox;Prox1CreERT2 mice were used to delete VE-cadherin in lymphatic endothelial cells across life stages, including embryonic, postnatal, and adult. Lymphatic architecture and function was characterized using immunostaining and functional lymphangiography. To evaluate the impact of temporal and functional regression of cardiac lymphatics in Cdh5flox/flox;Prox1CreERT2 mice, left anterior descending artery ligation was performed and cardiac function and repair after myocardial infarction was evaluated by echocardiography and histology. Cellular effects of VE-cadherin deletion on lymphatic signaling pathways were assessed by knockdown of VE-cadherin in cultured lymphatic endothelial cells. RESULTS: Embryonic deletion of VE-cadherin produced edematous embryos with dilated cardiac lymphatics with significantly altered vessel tip morphology. Postnatal deletion of VE-cadherin caused complete disassembly of cardiac lymphatics. Adult deletion caused a temporal regression of the quiescent epicardial lymphatic network which correlated with significant dermal and cardiac lymphatic dysfunction, as measured by fluorescent and quantum dot lymphangiography, respectively. Surprisingly, despite regression of cardiac lymphatics, Cdh5flox/flox;Prox1CreERT2 mice exhibited preserved cardiac function, both at baseline and following myocardial infarction, compared with control mice. Mechanistically, loss of VE-cadherin leads to aberrant cellular internalization of VEGFR3, precluding the ability of VEGFR3 to be either canonically activated by VEGF-C or noncanonically transactivated by adrenomedullin signaling, impairing downstream processes such as cellular proliferation. CONCLUSIONS: VE-cadherin is an essential scaffolding protein to maintain prolymphangiogenic signaling nodes at the plasma membrane, which are required for the development and adult maintenance of cardiac lymphatics, but not for cardiac function basally or after injury.

The nuclear receptor RORα preserves cardiomyocyte mitochondrial function by regulating caveolin-3-mediated mitophagy.

Preserving optimal mitochondrial function is critical in the heart, which is the most ATP-avid organ in the body. Recently, we showed that global deficiency of the nuclear receptor RORα in the "staggerer" mouse exacerbates angiotensin II-induced cardiac hypertrophy and compromises cardiomyocyte mitochondrial function. However, the mechanisms underlying these observations have not been defined previously. Here, we used pharmacological and genetic gain- and loss-of-function tools to demonstrate that RORα regulates cardiomyocyte mitophagy to preserve mitochondrial abundance and function. We found that cardiomyocyte mitochondria in staggerer mice with lack of functional RORα were less numerous and exhibited fewer mitophagy events than those in WT controls. The hearts of our novel cardiomyocyte-specific RORα KO mouse line demonstrated impaired contractile function, enhanced oxidative stress, increased apoptosis, and reduced autophagic flux relative to Cre(-) littermates. We found that cardiomyocyte mitochondria in "staggerer" mice with lack of functional RORα were upregulated by hypoxia, a classical inducer of mitophagy. The loss of RORα blunted mitophagy and broadly compromised mitochondrial function in normoxic and hypoxic conditions in vivo and in vitro. We also show that RORα is a direct transcriptional regulator of the mitophagy mediator caveolin-3 in cardiomyocytes and that enhanced expression of RORα increases caveolin-3 abundance and enhances mitophagy. Finally, knockdown of RORα impairs cardiomyocyte mitophagy, compromises mitochondrial function, and induces apoptosis, but these defects could be rescued by caveolin-3 overexpression. Collectively, these findings reveal a novel role for RORα in regulating mitophagy through caveolin-3 and expand our currently limited understanding of the mechanisms underlying RORα-mediated cardioprotection.

Decreased inspired oxygen stimulates de novo formation of coronary collaterals in adult heart.

RATIONALE: Collateral vessels lessen myocardial ischemia when acute or chronic coronary obstruction occurs. It has long been assumed that although native (pre-existing) collaterals enlarge in obstructive disease, new collaterals do not form in the adult. However, the latter was recently shown to occur after coronary artery ligation. Understanding the signals that drive this process is challenged by the difficulty in studying collateral vessels directly and the complex milieu of signaling pathways, including cell death, induced by ligation. Herein we show that hypoxemia alone is capable of inducing collateral vessels to form and that the novel gene Rabep2 is required. OBJECTIVE: Hypoxia stimulates angiogenesis during embryonic development and in pathological states. We hypothesized that hypoxia also stimulates collateral formation in adult heart by a process that involves RABEP2, a recently identified protein required for formation of collateral vessels during development. METHODS AND RESULTS: Exposure of mice to reduced FiO2 induced collateral formation that resulted in smaller infarctions following LAD ligation and that reversed on return to normoxia. Deletion of Rabep2 or knockdown of Vegfa inhibited formation. Hypoxia upregulated Rabep2, Vegfa and Vegfr2 in heart and brain microvascular endothelial cells (HBMVECs). Knockdown of Rabep2 impaired migration of HBMVECs. In contrast to systemic hypoxia, deletion of Rabep2 did not affect collateral formation induced by ischemic injury caused by LAD ligation. CONCLUSIONS: Hypoxia induced formation of coronary collaterals by a process that required VEGFA and RABEP2, proteins also required for collateral formation during development. Knockdown of Rabep2 impaired cell migration, providing one potential mechanism for RABEP2's role in collateral formation. This appears specific to hypoxia, since formation after acute ischemic injury was unaffected in Rabep2-/- mice. These findings provide a novel model for studying coronary collateral formation, and demonstrate that hypoxia alone can induce new collaterals to form in adult heart.

On the Bioactivity and Mechanical Properties of Gehlenite Nanobioceramic: A Comparative Study.

BACKGROUND: For a new biomaterial which is going to be applied in bone tissue regeneration, bioactivity (bone bonding ability) and desirable mechanical properties are very essential parameters to take into consideration. In the present study, the gehlenite's mechanical properties and bioactivity are assessed and compared with hydroxyapatite (HA) for bone tissue regeneration. METHOD: Gehlenite and HA nanoparticles are synthesized through sol-gel method and coprecipitation technique, respectively, and their physical and chemical properties are characterized through X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy. RESULTS: The results prove that the gehlenite and HA phases without any undesirable phase are obtained, and the particles of both compounds are in the nanometer range with spherical morphology. The compressive strength of both compounds are assessed, and the values for gehlenite and HA disks are 144 ± 5 and 150 ± 4.8 MPa, respectively. Next, their bioactivity potential is assessed into simulated body fluid (SBF) up to 21 days, and the results show that after 14 days, gehlenite disk's surface is completely covered with newly formed Ca-P particles. However, some sporadic precipitations after 21 days soaking into SBF are formed onto the HA disk's surface. CONCLUSION: This comparative study shows that nanostructured gehlenite disk with desirable mechanical properties and faster bioactivity kinetic than HA can be considered as a promising bioceramic for bone tissue regeneration.

Hypoxia induces de novo formation of cerebral collaterals and lessens the severity of ischemic stroke.

Pial collaterals provide protection in stroke. Evidence suggests their formation late during gestation (collaterogenesis) is driven by reduced oxygen levels in the cerebral watersheds. The purpose of this study was to determine if collaterogenesis can be re-activated in the adult to induce formation of additional collaterals ("neo-collateral formation", NCF). Mice were gradually acclimated to reduced inspired oxygen (FIO2) and maintained at 12, 10, 8.5 or 7% for two-to-eight weeks. Hypoxemia induced "dose"-dependent NCF and remodeling of native collaterals, and decreased infarct volume after permanent MCA occlusion. In contrast, no formation occurred of addition collateral-like intra-tree anastomoses, PComs, or branches within the MCA tree. Hypoxic NCF, remodeling and infarct protection were durable, i.e. retained for at least six weeks after return to normoxia. Hypoxia increased expression of Hif2α, Vegfa, Rabep2, Angpt2, Tie2 and Cxcr4. Neo-collateral formation was abolished in mice lacking Rabep2, a novel gene involved in VEGFA→Flk1 signaling and required for formation of collaterals during development, and inhibited by knockdown of Vegfa, Flk1 and Cxcr4. Rabep2-dependent NCF was also induced by permanent MCA occlusion. This is the first report that hypoxia induces new pial collaterals to form. Hypoxia- and occlusion-induced neo-collateral formation provide models to study collaterogenesis in the adult.

Hierarchical porous Mg2SiO4-CoFe2O4 nanomagnetic scaffold for bone cancer therapy and regeneration: Surface modification and in vitro studies.

3D multifunctional bone scaffolds have recently attracted more attention in bone tissue engineering because of addressing critical issues like bone cancer and inflammation beside bone regeneration. In this study, a 3D bone scaffold is fabricated from Mg2SiO4-CoFe2O4 nanocomposite which is synthesized via a two-step synthesis strategy and then the scaffold's surface is modified with poly-3-hydroxybutyrate (P3HB)-ordered mesoporous magnesium silicate (OMMS) composite to improve its physicochemical and biological properties. The Mg2SiO4-CoFe2O4 scaffold is fabricated through polymer sponge technique and the scaffold exhibits an interconnected porous structure in the range of 100-600 µm. The scaffold is then coated with OMMS/P3HB composite via dip coating and the physical, chemical, and biological-related properties of OMMS/P3HB composite-coated scaffold are assessed and compared to the non-coated and P3HB-coated scaffolds in vitro. It is found that, on the one hand, P3HB increases the cell attachment, proliferation, and compressive strength of the scaffold, but on the other hand, it weakens the bioactivity kinetic. Addition of OMMS to the coating composition is accompanied with significant increase in bioactivity kinetic. Besides, OMMS/P3HB composite-coated scaffold exhibits higher drug loading capacity and more controlled release manner up to 240 h than the other samples because of OMMS which has a high surface area and ordered mesoporous structure suitable for controlled release applications. The overall results indicate that OMMS/P3HB coating on Mg2SiO4-CoFe2O4 scaffold leads to a great improvement in bioactivity, drug delivery potential, compressive strength, cell viability, and proliferation. Moreover, OMMS/P3HB composite-coated scaffold has heat generation capability for hyperthermia-based bone cancer therapy and so it is suggested as a multifunctional scaffold with great potentials for bone cancer therapy and regeneration.

Nanostructured magnetic Mg2SiO4-CoFe2O4 composite scaffold with multiple capabilities for bone tissue regeneration.

Multifunctional magnetic 3D scaffolds are recently of particular interest because of their applications in hyperthermia-based therapy and localized drug delivery beside of their basic properties to be applied in bone tissue regeneration. In the current study, a magnetic nanocomposite is designed and synthesized through a two-step synthesis strategy in which CoFe2O4 nanoparticles are prepared via sol-gel combustion method and then they are coated through sol-gel method with Mg2SiO4. The characterization relates to the nanocomposite shows that Mg2SiO4-CoFe2O4 is successfully synthesized and it has a core-shell structure. Then, 3D scaffolds are fabricated through polymer sponge technique from the nanocomposite. Physiochemical and biological properties of the scaffolds are assessed in vitro amongst which bioactivity, biodegradability, mechanical properties, hyperthermia capability, controlled release potential, antibacterial activity, cell compatibility and attachment can be mentioned. The results demonstrate that the scaffolds have high porous structure with interconnected porosity and desirable mechanical properties close to cancellous bone. The magnetic scaffold is biodegradable and bioactive and exhibits controlled release of rifampin as an antibiotic drug up to 96 h. Moreover, in the exposure of different magnetic fields it has potential to produce heat for different kinds of hyperthermia-based therapies. The antibacterial activity of drug-loaded scaffold is assessed against S. aureus bacteria. The results suggest that Mg2SiO4-CoFe2O4 nanocomposite scaffold with multiple capabilities has a great potential to be applied in the case of large bone defects which are caused by tumors to not only eradicate remained cancerous tissues, but also prevent infection after surgery and regenerate bone defect.

Cerebral Collateral Circulation: A Review in the Context of Ischemic Stroke and Mechanical Thrombectomy.

The pial (leptomenigeal) collateral circulation is a key determinant of functional outcome after mechanical thrombectomy after large-vessel ischemic stroke. Patients with good collateral blood flow benefit up to 24 hours after stroke onset, whereas those with poor collateral flow evidence less or no benefit. However, clues to why collateral flow varies so widely among patients have remained elusive. Recent findings in animal studies, which are currently being tested for confirmation in humans, have found that naturally occurring variants of a novel "collateral gene," Rabep2, result in large differences in the extent of anatomic collaterals and thus blood flow and infarct size in mice after stroke. The comprehension of collagerogenesis in humans and the evaluation of collateral status could aid in identifying patients who will benefit not only from mechanical thrombectomy in the extended time window but also from any reperfusion strategy. We performed a literature review focused on radiographic, clinical, and genetic aspects of the collateral circulation.

Accurate quantification of brown adipose tissue mass by xenon-enhanced computed tomography.

Detection and quantification of brown adipose tissue (BAT) mass remains a major challenge, as current tomographic imaging techniques are either nonspecific or lack the necessary resolution to quantify BAT mass, especially in obese phenotypes, in which this tissue may be present but inactive. Here, we report quantification of BAT mass by xenon-enhanced computed tomography. We show that, during stimulation of BAT thermogenesis, the lipophilic gas xenon preferentially accumulates in BAT, leading to a radiodensity enhancement comparable to that seen in the lungs. This enhancement is mediated by a selective reduction in BAT vascular resistance, which greatly increases vascular perfusion of BAT. This enhancement enables precise identification and quantification of BAT mass not only in lean, but also in obese, mouse phenotypes, in which this tissue is invisible to conventional tomographic imaging techniques. The method is developed and validated in rodents and then applied in macaques to assess its feasibility in larger species.

Sex Differences in the Cerebral Collateral Circulation.

Premenopausal women and intact female rodents sustain smaller cerebral infarctions than males. Several sex-dependent differences have been identified as potential contributors, but many questions remain unanswered. Mice exhibit wide variation in native collateral number and diameter (collateral extent) that is dependent on differences in genetic background, aging, and other comorbidities and that contributes to their also-wide differences in infarct volume. Likewise, variation in infarct volume correlates with differences in collateral-dependent blood flow in patients with acute ischemic stroke. We examined whether extent of pial collateral arterioles and posterior communicating collateral arteries (PComAs) differ depending on sex in young, aged, obese, hypertensive, and genetically different mice. We combined new data with meta-analysis of our previously published data. Females of C57BL/6J (B6) and BALB/cByJ (BC) strains sustained smaller infarctions than males after permanent MCA occlusion. This protection was unchanged in BC mice after introgression of the B6 allele of Dce1, the major genetic determinant of variation in pial collaterals among mouse strains. Consistent with this, collateral extent in these and other strains did not differ with sex. Extent of PComAs and primary cerebral arteries also did not vary with sex. No dimorphism was evident for loss of pial collateral number and/or diameter (collateral rarefaction) caused by aging, obesity, and hypertension, nor for collateral remodeling after pMCAO. However, rarefaction was greater in females with long-standing hypertension. We conclude that smaller infarct volume in female mice is not due to greater collateral extent, greater remodeling, or less rarefaction caused by aging, obesity, or hypertension.

Redox regulation of Ras and Rho GTPases: mechanism and function.

SIGNIFICANCE: Oxidation and reduction events are critical to physiological and pathological processes and are highly regulated. Herein, we present evidence for the role of Ras and Rho GTPases in controlling these events and the unique underlying mechanisms. Evidence for redox regulation of Ras GTPases that contain a redox-sensitive cysteine (X) in the conserved NKXD motif is presented, and a growing consensus supports regulation by a thiyl radical-mediated oxidation mechanism. We also discuss the debate within the literature regarding whether 2e(-) oxidation mechanisms also regulate Ras GTPase activity. RECENT ADVANCES: We examine the increasing in vitro and cell-based data supporting oxidant-mediated activation of Rho GTPases that contain a redox-sensitive cysteine at the end of the conserved phosphoryl-binding loop (p-loop) motif (GXXXXG[S/T]C). While this motif is distinct from Ras, these data suggest a similar 1e(-) oxidation-mediated activation mechanism. CRITICAL ISSUES: We also review the data showing that the unique p-loop placement of the redox-sensitive cysteine in Rho GTPases supports activation by 2e(-) cysteine oxidation. Finally, we examine the role that Ras and Rho GTPases play in controlling key oxidant-regulating enzymes in the cell, and we speculate on a feedback mechanism. FUTURE DIRECTIONS: Given that these GTPases and redox-regulating enzymes are involved in multiple physiological and pathological processes, we discuss future experiments that may clarify the interplay between them.

Isoform-specific differences between Rap1A and Rap1B GTPases in the formation of endothelial cell junctions.

Rap1 is a Ras-like GTPase that has been studied with respect to its role in cadherin-based cell adhesion. Rap1 exists as two separate isoforms, Rap1A and Rap1B, which are 95% identical and yet the phenotype of the isoform-specific knockout mice is different. We and others have previously identified a role for Rap1 in regulating endothelial adhesion, junctional integrity and barrier function; however, these early studies did not distinguish a relative role for each isoform. To dissect the individual contribution of each isoform in regulating the endothelial barrier, we utilized an engineered microRNA-based approach to silence Rap1A, Rap1B or both, then analyzed barrier properties of the endothelium. Electrical impedance sensing experiments show that Rap1A is the predominant isoform involved in endothelial cell junction formation. Quantification of monolayer integrity by VE-cadherin staining revealed that knockdown of Rap1A, but not Rap1B, increased the number of gaps in the confluent monolayer. This loss of monolayer integrity could be rescued by re-expression of exogenous Rap1A protein. Expression of GFP-tagged Rap1A or 1B revealed quantifiable differences in localization of each isoform, with the junctional pool of Rap1A being greater. The junctional protein AF-6 also co-immunoprecipitates more strongly with expressed GFP-Rap1A. Our results show that Rap1A is the more critical isoform in the context of endothelial barrier function, indicating that some cellular processes differentially utilize Rap1A and 1B isoforms. Studying how Rap1 isoforms differentially regulate EC junctions may thus reveal new targets for developing therapeutic strategies during pathological situations where endothelial barrier disruption leads to disease.

Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif.

BACKGROUND: Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs. CONCLUSIONS/SIGNIFICANCE: Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.