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The dura, arachnoid and pia mater, as the constituent layers of the meninges, along with cerebrospinal fluid in the subarachnoid space and ventricles, are essential protectors of the brain and spinal cord. Complemented by immune cells, blood vessels, lymphatic vessels and nerves, these connective tissue layers have held many secrets that have only recently begun to be revealed. Each meningeal layer is now known to have molecularly distinct types of fibroblasts. Cerebrospinal fluid clearance through peripheral lymphatics and lymph nodes is well documented, but its routes and flow dynamics are debated. Advances made in meningeal immune functions are also debated. This Review considers the cellular and molecular structure and function of the dura, arachnoid and pia mater in the context of conventional views, recent progress, and what is uncertain or unknown. The hallmarks of meningeal pathophysiology are identified toward developing a more complete understanding of the meninges in health and disease.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1–17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFPlymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals. RESEARCH DESIGN AND METHODS--Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days -3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test. RESULTS--Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of [p47.sup.phox] and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1-treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin. CONCLUSIONS--These findings support the concept of cavernous endothelial regeneration by use of the recombinant Ang1 protein as a curative therapy for diabetic erectile dysfunction., Erectile dysfunction affects up to 75% of all men with diabetes and occurs earlier in such patients than in the general population (1,2). The Massachusetts Male Aging Study revealed that [...]

Endothelial cells (ECs) adapt their metabolism to enable the growth of new blood vessels, but little is known how ECs regulate metabolism to adopt a quiescent state. Here, we show that the metabolite S-2-hydroxyglutarate (S-2HG) plays a crucial role in the regulation of endothelial quiescence. We find that S-2HG is produced in ECs after activation of the transcription factor forkhead box O1 (FOXO1), where it limits cell cycle progression, metabolic activity and vascular expansion. FOXO1 stimulates S-2HG production by inhibiting the mitochondrial enzyme 2-oxoglutarate dehydrogenase. This inhibition relies on branched-chain amino acid catabolites such as 3-methyl-2-oxovalerate, which increase in ECs with activated FOXO1. Treatment of ECs with 3-methyl-2-oxovalerate elicits S-2HG production and suppresses proliferation, causing vascular rarefaction in mice. Our findings identify a metabolic programme that promotes the acquisition of a quiescent endothelial state and highlight the role of metabolites as signalling molecules in the endothelium.

Meningeal lymphatics are conduits for cerebrospinal fluid drainage to lymphatics and lymph nodes in the neck. In this issue of JEM, Boisserand et al. (https://doi.org/10.1084/jem.20221983) provide evidence that expansion of meningeal lymphatics protects against ischemic stroke.

The four-chamber structure of the mammalian heart is established during embryonic development. While key regulators for ventricular development are well studied, regulatory mechanisms for atrial chamber morphogenesis remain poorly understood. Here, we found that angiopoietin-1 (Angpt1), a vascular maturation factor, is highly and specifically expressed in atrial myocardium during heart development. Loss of myocardial Angpt1 in mouse embryo led to severe impairment in atrial chamber morphogenesis. We revealed that Angpt1 deficiency results in excessive deposition of cardiac jelly, which disturbs regulation of myocardial growth, thereby impairing maturation of atrial chambers. Mechanistically, myocardial Angpt1 activates endocardial Tie2 and positively regulates expression of ADAMTS proteases, which is crucial for proper degradation of cardiac jelly. Accordingly, loss of Tie2 also impairs ADAMTS-mediated degradation of cardiac jelly in atrium. Collectively, myocardial Angpt1/endocardial Tie2 signaling in atrium promotes spatiotemporal degradation of cardiac jelly during early cardiac development and is therefore indispensable for atrial chamber morphogenesis.

High-grade glioma (HGG) is highly angiogenic, but antiangiogenic therapy has transient clinical benefit in only a fraction of patients. Vascular regulators of these heterogeneous responses remain undetermined. We found up-regulation of Sox7 and down-regulation of Sox17 in tumor endothelial cells (tECs) in mouse HGG. Sox7 deletion suppressed VEGFR2 expression, vascular abnormality, hypoxia-driven invasion, regulatory T cell infiltration, and tumor growth. Conversely, Sox17 deletion exacerbated these phenotypes by up-regulating Sox7 in tECs. Anti-VEGFR2 antibody treatment delayed tumor growth by normalizing Sox17-deficient abnormal vessels with high Sox7 levels but promoted it by regressing Sox7-deficient vessels, recapitulating variable therapeutic responses to antiangiogenic therapy in HGG patients. Our findings establish that Sox7 promotes tumor growth via vessel abnormalization, and its level determines the therapeutic outcome of VEGFR2 inhibition in HGG. In 189 HGG patients, Sox7 expression was heterogeneous in tumor vessels, and high Sox7 levels correlated with poor survival, early recurrence, and impaired vascular function, emphasizing the clinical relevance of Sox7 in HGG.

Recent discoveries of novel functions and diverse origins of lymphatic vessels have drastically changed our view of lymphatic vasculature. Traditionally regarded as passive conduits for fluid and immune cells, lymphatic vessels now emerge as active, tissue-specific players in major physiological and pathophysiological processes. Lymphatic vessels show remarkable plasticity and heterogeneity, reflecting their functional specialization to control the tissue microenvironment. Moreover, alternative developmental origins of lymphatic endothelial cells in some organs may contribute to the diversity of their functions in adult tissues. This review aims to summarize the most recent findings of organotypic differentiation of lymphatic endothelial cells in terms of their distinct (patho)physiological functions in skin, lymph nodes, small intestine, brain, and eye. We discuss recent advances in our understanding of the heterogeneity of lymphatic vessels with respect to the organ-specific functional and molecular specialization of lymphatic endothelium, such as the hybrid blood-lymphatic identity of Schlemm’s canal, functions of intestinal lymphatics in dietary fat uptake, and discovery of meningeal lymphatic vasculature and perivascular brain lymphatic endothelial cells.

Vascular endothelial (VE)–protein tyrosine phosphatase (PTP) associates with VE-cadherin, thereby supporting its adhesive activity and endothelial junction integrity. VE-PTP also associates with Tie-2, dampening the tyrosine kinase activity of this receptor that can support stabilization of endothelial junctions. Here, we have analyzed how interference with VE-PTP affects the stability of endothelial junctions in vivo. Blocking VE-PTP by antibodies, a specific pharmacological inhibitor (AKB-9778), and gene ablation counteracted vascular leak induction by inflammatory mediators. In addition, leukocyte transmigration through the endothelial barrier was attenuated. Interference with Tie-2 expression in vivo reversed junction-stabilizing effects of AKB-9778 into junction-destabilizing effects. Furthermore, lack of Tie-2 was sufficient to weaken the vessel barrier. Mechanistically, inhibition of VE-PTP stabilized endothelial junctions via Tie-2, which triggered activation of Rap1, which then caused the dissolution of radial stress fibers via Rac1 and suppression of nonmuscle myosin II. Remarkably, VE-cadherin gene ablation did not abolish the junction-stabilizing effect of the VE-PTP inhibitor. Collectively, we conclude that inhibition of VE-PTP stabilizes challenged endothelial junctions in vivo via Tie-2 by a VE-cadherin–independent mechanism. In the absence of Tie-2, however, VE-PTP inhibition destabilizes endothelial barrier integrity in agreement with the VE-cadherin–supportive effect of VE-PTP.

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To unveil the organotypic role and vulnerability of lymphatic vessels, we generated a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-Cre/iDTR double-transgenic mouse and ablated LYVE-1–expressing lymphatic vessels in adult mice in a diphtheria toxin (DT)–inducible manner based on selective expression of LYVE-1 in most lymphatic vessels. Strikingly, lymphatic vessels in the small intestine and lymph nodes were rapidly ablated, but lymphatic vessels in the other organs were relatively intact at 24 hours after DT administration. Unexpectedly, LYVE-1-Cre/iDTR mice died of sepsis without visible edema at 24 and 60 hours after DT administration. The cause of death appeared to be related to acute failure of immune surveillance systems in the small intestine and draining lymph nodes. Of note, acute loss of lymphatic lacteals in intestinal villi appeared to trigger distortion of blood capillaries and the whole architecture of the villi, whereas acute loss of lymphatic vessels in lymph nodes caused dysfunction of lymph drainage and abnormal distribution of dendritic cells and macrophages. Thus, intact lymphatic vessels are required for structural and functional maintenance of surrounding tissues in an organotypic manner, at least in the intestine and lymph nodes.

Successful differentiation and expansion of endothelial cells (ECs) from embryonic stem cell (ESC)–derived Flk1+ mesodermal precursor cells (MPCs) requires supplementation of vascular endothelial growth factor-A (VEGF-A). While analyzing VEGF-A/VEGFR2 downstream signaling pathway that underlies the VEGF-A-induced differentiation and expansion of ECs, we fortuitously found that Rho-associated protein kinase (ROCK) inhibitor Y27632 profoundly promoted the differentiation and expansion of ECs from Flk1+ MPCs while reducing the differentiation and expansion of mural cells. The ROCK suppression-induced expansion of ECs appears to have resulted from promotion of proliferation of ECs via activation of PI3-kinase-Akt signaling. The ECs obtained by the combination of ROCK suppression and VEGF-A supplementation faithfully expressed most pan-EC surface makers, and phenotypic analyses revealed that they were differentiated toward arterial EC. Further incubation of the ICAM2+ ECs with Y27632 and VEGF-A for 2 days promoted expansion of ECs by 6.5-fold compared with those incubated with only VEGF-A. Importantly, the ROCK suppression-induced ECs displayed neovasculogenic abilities in vitro and in vivo. Thus, supplementation of ROCK inhibitor Y27632 along with VEGF-A in 2D Matrigel culture system provides a simple, efficient, and versatile method for obtaining ample amount of ESC-derived ECs at high purity suitable for use in therapeutic neovascularization.

Successful differentiation and expansion of endothelial cells (ECs) from embryonic stem cell (ESC)–derived Flk1+mesodermal precursor cells (MPCs) requires supplementation of vascular endothelial growth factor-A (VEGF-A). While analyzing VEGF-A/VEGFR2 downstream signaling pathway that underlies the VEGF-A-induced differentiation and expansion of ECs, we fortuitously found that Rho-associated protein kinase (ROCK) inhibitor Y27632 profoundly promoted the differentiation and expansion of ECs from Flk1+MPCs while reducing the differentiation and expansion of mural cells. The ROCK suppression-induced expansion of ECs appears to have resulted from promotion of proliferation of ECs via activation of PI3-kinase-Akt signaling. The ECs obtained by the combination of ROCK suppression and VEGF-A supplementation faithfully expressed most pan-EC surface makers, and phenotypic analyses revealed that they were differentiated toward arterial EC. Further incubation of the ICAM2+ECs with Y27632 and VEGF-A for 2 days promoted expansion of ECs by 6.5-fold compared with those incubated with only VEGF-A. Importantly, the ROCK suppression-induced ECs displayed neovasculogenic abilities in vitro and in vivo. Thus, supplementation of ROCK inhibitor Y27632 along with VEGF-A in 2D Matrigel culture system provides a simple, efficient, and versatile method for obtaining ample amount of ESC-derived ECs at high purity suitable for use in therapeutic neovascularization.

Angiopoietin-1 (Ang1) plays a crucial role in vascular and hematopoietic development, mainly through its cognate receptor Tie2. However, little is known about the precise role of Ang1 in embryonic stem cell (ESC) differentiation. In the present study, we used COMP-Ang1 (a soluble and potent variant of Ang1) to explore the effect of Ang1 on endothelial and hematopoietic differentiation of mouse ESCs in an OP9 coculture system and found that Ang1 promoted endothelial cell (EC) differentiation from Flk-1+mesodermal precursors. This effect mainly occurred through Tie2 signaling and was altered in the presence of soluble Tie2-Fc. We accounted for this Ang1-induced expansion of ECs as enhanced proliferation and survival. Ang1 also had an effect on CD41+cells, transient precursors that can differentiate into both endothelial and hematopoietic lineages. Intriguingly, Ang1 induced the preferential differentiation of CD41+cells toward ECs instead of hematopoietic cells. This EC expansion promoted by Ang1 was also recapitulated in induced pluripotent stem cells (iPSCs) and human ESCs. We successfully achieved in vivo neovascularization in mice by transplantation of ECs obtained from Ang1-stimulated ESCs. We conclude that Ang1/Tie2 signaling has a pivotal role in ESC-EC differentiation and that this effect can be exploited to expand EC populations.

Blood vessel leakiness is an early, transient event in acute inflammation but can also persist as vessels undergo remodeling in sustained inflammation. Angiopoietin/Tie2 signaling can reduce the leakiness through changes in endothelial cells. The role of pericytes in this action has been unknown. We used the selective PDGF-B-blocking oligonucleotide aptamer AX102 to determine whether disruption of pericyte-endothelial crosstalk alters vascular leakiness or remodeling in the airways of mice under four different conditions: i) baseline, ii) acute inflammation induced by bradykinin, iii) sustained inflammation after 7-day infection by the respiratory pathogen Mycoplasma pulmonis, or iv) leakage after bradykinin challenge in the presence of vascular stabilization by the angiopoietin-1 (Ang1) mimic COMP-Ang1 for 7 days. AX102 reduced pericyte coverage but did not alter the leakage of microspheres from tracheal blood vessels at baseline or after bradykinin; however, AX102 exaggerated leakage at 7 days after M. pulmonisinfection and increased vascular remodeling and disease severity at 14 days. AX102 also abolished the antileakage effect of COMP-Ang1 at 7 days. Together, these findings show that pericyte contributions to endothelial stability have greater dependence on PDGF-B during the development of sustained inflammation, when pericyte dynamics accompany vascular remodeling, than under baseline conditions or in acute inflammation. The findings also show that the antileakage action of Ang1 requires PDGF-dependent actions of pericytes in maintaining endothelial stability.

Recruitment and adhesion of bone marrow (BM)–derived circulating progenitor cells to ischemic tissue are important for vasculogenesis and tissue repair. Recently, we found cartilage oligomeric matrix protein (COMP)–Ang1 is a useful cell-priming agent to improve the therapeutic efficacy of progenitor cells. However, the effect and the underlying mechanisms of COMP-Ang1 on recruitment of BM-derived progenitor cells (BMPCs) to foci of vascular injury have not been well defined. Here, we found that COMP-Ang1 is a critical stimulator of stromal cell–derived factor 1 (SDF-1), the principal regulator of BM-cell trafficking. Furthermore, SDF-1 stimulation by COMP-Ang1 was blocked by small-interfering RNA (siRNA) against hypoxia-inducible factor-1α (HIF-1α). COMP-Ang1 increased the synthesis of HIF-1α by activating mammalian target of rapamycin (mTOR) in hypoxic endothelium. The intermediate mechanism transmitting the COMP-Ang1 signal to the downstream mTOR/HIF-1α/SDF-1 pathway was the enhanced binding of the Tie2 receptor with integrin-linked kinase (ILK), an upstream activator of mTOR. In the mouse ischemic model, local injection of COMP-Ang1 stimulated the incorporation of BMPCs into ischemic limb, thereby enhancing neovasculogenesis and limb salvage. Collectively, our findings identify the COMP-Ang1/HIF-1α/SDF-1 pathway as a novel inducer of BMPC recruitment and neovasculogenesis in ischemic disease.

The stromal vascular fraction (SVF) in adipose tissue contains a pool of various stem and progenitor cells, but the existence of hematopoietic stem and progenitor cells (HSPCs) in the SVF has not been seriously considered. We detected the presence of HSPCs in the SVF by phenotypically probing with Lin−Sca-1+c-kit+ (LSK) and functionally confirming the presence using colony-forming cell assay and assessing the long-term multilineage reconstitution ability after SVF transplantation. The LSK population in the SVF was 0.004% plus or minus 0.001%, and 5 × 105 freshly isolated SVF cells gave rise to 13 plus or minus 4 multilineage colonies. In addition, 0.15% plus or minus 0.03% of SVF cells was home to bone marrow (BM), especially near vascular and endosteal regions, 24 hours after blood transplantation. SVF transplantation was capable of generating a long-term (> 16 weeks), but variable extent (2.1%-32.1%) multilineage reconstitution in primary recipients, which was subsequently transferred to the secondary recipients by BM transplantation. All HSPCs within the SVF originated from the BM. Furthermore, the granulocyte–colony-stimulating factor (G-CSF) mobilization of HSPCs from BM markedly elevated the number of phenotypic and functional HSPCs in the SVF, which induced a high efficiency long-term reconstitution in multilineage hematopoiesis in vivo. Our results provide compelling evidence that adipose tissue is a novel extramedullary tissue possessing phenotypic and functional HSPCs.

We sought to determine the effects of activation of peroxisome proliferator-activated receptor-? (PPAR-?) on multilocularization of adipocytes in adult white adipose tissue (WAT). Male C57BL/6 normal, db/db, and ob/ob mice were treated with agonists of PPAR-?, PPAR-a, or ß3-adrenoceptor for 3 weeks. To distinguish multilocular adipocytes from unilocular adipocytes, whole-mounted adipose tissues were co-immunostained for perilipin and collagen IV. PPAR-? activation with rosiglitazone or pioglitazone induced a profound change of unilocular adipocytes into smaller, multilocular adipocytes in adult WAT in a time-dependent, dose-dependent, and reversible manner. PPAR-a activation with fenofibrate did not affect the number of locules or remodeling. db/db and ob/ob obese mice exhibited less multilocularization in response to PPAR-? activation compared to normal mice. Nevertheless, all adipocytes activated by PPAR-? contained a single nucleus regardless of locule number. Multilocular adipocytes induced by PPAR-? activation contained substantially increased mitochondrial content and enhanced expression of uncoupling protein-1, PPAR-? coactivator-1-a , and perilipin. Taken together, PPAR-? activation induces profound multilocularization and enhanced mitochondrial biogenesis in the adipocytes of adult WAT. These changes may affect the overall function of WAT.

Lymphatic vessels in the diaphragm are essential for draining peritoneal fluid, but little is known about their pathological changes during inflammation. Here we characterized diaphragmatic lymphatic vessels in a peritonitis model generated by daily i.p. administration of lipopolysaccharide (LPS) in mice. Intraperitoneal LPS increased lymphatic density, branching, sprouts, connections, and network formation in the diaphragm in time- and dose-dependent manners. These changes were reversible on discontinuation of LPS administration. The LPS-induced lymphatic density and remodeling occur mainly through proliferation of lymphatic endothelial cells. CD11b+macrophages were massively accumulated and closely associated with the lymphatic vessels changed by i.p. LPS. Both RT-PCR assays and experiments with vascular endothelial growth factor-C/D blockade and macrophage-depletion indicated that the CD11b+macrophage-derived lymphangiogenic factors vascular endothelial growth factor-C/D could be major mediators of LPS-induced lymphangiogenesis and lymphatic remodeling through paracrine activity. Functional assays with India ink and fluorescein isothiocyanate-microspheres indicated that impaired peritoneal fluid drainage in diaphragm of LPS-induced peritonitis mice was due to inflammatory fibrosis and massive attachment of CD11b+macrophages on the peritoneal side of the diaphragmatic lymphatic vessels. These findings reveal that CD11b+macrophages play an important role in i.p. LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm.

Using a bacterial pathogen–induced acute inflammation model in the skin, we defined the roles of local lymphatic vessels and draining lymph nodes (DLNs) in antigen clearance and inflammation resolution. At the peak day of inflammation, robust expansion of lymphatic vessels and profound infiltration of CD11b+/Gr-1+macrophages into the inflamed skin and DLN were observed. Moreover, lymph flow and inflammatory cell migration from the inflamed skin to DLNs were enhanced. Concomitantly, the expression of lymphangiogenic growth factors such as vascular endothelial growth factor C (VEGF-C), VEGF-D, and VEGF-A were significantly up-regulated in the inflamed skin, DLNs, and particularly in enriched CD11b+macrophages from the DLNs. Depletion of macrophages, or blockade of VEGF-C/D or VEGF-A, largely attenuated these phenomena, and produced notably delayed antigen clearance and inflammation resolution. Conversely, keratin 14 (K14)–VEGF-C transgenic mice, which have dense and enlarged lymphatic vessels in the skin dermis, exhibited accelerated migration of inflammatory cells from the inflamed skin to the DLNs and faster antigen clearance and inflammation resolution. Taken together, these results indicate that VEGF-C, -D, and -A derived from the CD11b+/Gr-1+macrophages and local inflamed tissues play a critical role in promoting antigen clearance and inflammation resolution.

Using a bacterial pathogen–induced acute inflammation model in the skin, we defined the roles of local lymphatic vessels and draining lymph nodes (DLNs) in antigen clearance and inflammation resolution. At the peak day of inflammation, robust expansion of lymphatic vessels and profound infiltration of CD11b+/Gr-1+ macrophages into the inflamed skin and DLN were observed. Moreover, lymph flow and inflammatory cell migration from the inflamed skin to DLNs were enhanced. Concomitantly, the expression of lymphangiogenic growth factors such as vascular endothelial growth factor C (VEGF-C), VEGF-D, and VEGF-A were significantly up-regulated in the inflamed skin, DLNs, and particularly in enriched CD11b+ macrophages from the DLNs. Depletion of macrophages, or blockade of VEGF-C/D or VEGF-A, largely attenuated these phenomena, and produced notably delayed antigen clearance and inflammation resolution. Conversely, keratin 14 (K14)–VEGF-C transgenic mice, which have dense and enlarged lymphatic vessels in the skin dermis, exhibited accelerated migration of inflammatory cells from the inflamed skin to the DLNs and faster antigen clearance and inflammation resolution. Taken together, these results indicate that VEGF-C, -D, and -A derived from the CD11b+/Gr-1+ macrophages and local inflamed tissues play a critical role in promoting antigen clearance and inflammation resolution.

The lymphatic vessel is a major conduit for immune cell transport; however, little is known about how lymphatic vessels regulate immune cell trafficking and how lymphatic vessels themselves respond to inflammation. Toll-like receptor 4 (TLR4) plays a central role in lipopolysaccharide (LPS)–induced inflammation, but the role of TLR4 in lymphatic endothelial cells (LECs) is poorly understood. Here, we found that LECs express high amounts of TLR4 in the intracellular region, and that the TLR4 of LECs is the main mediator of nuclear factor–κB (NF-κB) activation by LPS. LPS-TLR4 signaling in LECs resulted in the production of various chemokines for chemotaxis of macrophage. In addition, TLR4 in LECs actively contributed to the recruitment of macrophages to the draining lymphatic vessel. Furthermore, the macrophages that infiltrated into the lymphatic vessel induced lymphangiogenesis by secreting lymphangiogenic growth factors. These phenomena were largely attenuated not only in the mice defective in TLR4 signaling but also in the chimeric mice defective in TLR4 signaling that were recipients for bone marrow transplantation from normal TLR4-signaling mice. In conclusion, TLR4 in LECs plays an essential role in LPS-induced inflammatory lymphangiogenesis by chemotactic recruitment of macrophages.

Reactive oxygen species (ROS) play a crucial role in acute lung injury. Tissue inflammation, the increased vascular permeability, and plasma exudation are cardinal features of acute lung injury. Angiopoietin-1 (Ang1) has potential therapeutic applications in preventing vascular leakage and also has beneficial effects in several inflammatory disorders. Recently developed COMP-Ang1 is more potent than native Ang1 in phosphorylating tyrosine kinase with immunoglobulin and EGF homology domain 2 receptor in endothelial cells. However, there are no data on effects and related molecular mechanisms of COMP-Ang1 on ROS-induced acute lung injury. We used hydrogen peroxide (H2O2)-inhaled mice to evaluate the effect of COMP-Ang1 on pulmonary inflammation, bronchial hyper-responsiveness, and vascular leakage in acute lung injury. The results have revealed that VEGF expression, the levels of IL-4, TNF-a, IL-1ß, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in lungs, the levels of hypoxia-inducible factor-1a (HIF-1a) and NF-?B in nuclear protein extracts, phosphorylation of Akt, and vascular permeability were increased after inhalation of H2O2and that the administration of COMP-Ang1 markedly reduced airway hyper-responsiveness, pulmonary inflammation, plasma extravasation, and the increases of cytokines, adhesion molecules, and VEGF in lungs treated with H2O2. We have also found that the activation of HIF-1a and NF-?B and the increase of phosphoinositide 3-kinase activity in lung tissues after H2O2inhalation were decreased by the administration of COMP-Ang1. These results suggest that COMP-Ang1 ameliorates ROS-induced acute lung injury through attenuating vascular leakage and modulating inflammatory mediators.

Angiopoietin-1 (Ang1) is a vasculogenic factor which is signaled through the endothelial and bone marrow cell-specific, Tie2 receptor tyrosine kinase and has potential therapeutic applications for the induction of angiogenesis, enhancing endothelial cell survival, and preventing vascular leakage. In this study, we examined whether Ang1 directly exhibits bone marrow protection after ionizing radiation (IR) using an adenoviral vector of COMP-Ang1 (Ad-COMP-Ang1). This is a variant of Ang1 by replacement of the N-terminal portion of Ang1 with short coiled-coil domains of cartilage oligomeric matrix protein-Angiopoietin 1 (COMP-Ang1) which are, long enough for oligomerization but short enough to avoid problems of aggregation and insolubility. A spleen colony assay after 4.5 Gy whole body radiation, indicated that COMP-Ang1 significantly increased the mean colony numbers. Both the decrease in bone marrow cellularity and increased TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) positive cells produced by radiation in bone marrow were significantly inhibited by COMP-Ang1 transfer. The expression of the ligands of Ang1 and Tie2 receptors were increased by radiation and, the COMP-Ang1 transfer potentiated this protein expression. Pre-treatment of Ang1 could be beneficial in protecting bone marrow from damage by radiation and COMP-Ang1 may be an effective alternative to native Ang1 for therapeutic purposes.

Inflammation of the asthmatic airway is usually accompanied by increased vascular permeability and plasma exudation. Angiopoietin-1 (Ang1) has potential therapeutic applications in preventing vascular leakage. Recently, we developed a soluble, stable, and potent Ang1 variant, COMP-Ang1. COMP-Ang1 is more potent than native Ang1 in phosphorylating the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 receptor in lung endothelial cells. We have used a mouse model for allergic airway disease to determine effects of COMP-Ang1 on allergen-induced bronchial inflammation and airway hyper-responsiveness. These mice develop the following typical pathophysiological features of allergic airway disease in the lungs: increased numbers of inflammatory cells of the airways, airway hyper-responsiveness, increased levels of Th2 cell cytokines (IL-4, IL-5, and IL-13), adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), and chemokines (eotaxin and RANTES), and increased vascular permeability. Intravenous administration of COMP-Ang1 reduced bronchial inflammation and airway hyper-responsiveness. In addition, the increased plasma extravasation in allergic airway disease was significantly reduced by the administration of COMP-Ang1. These results suggest that COMP-Ang1 attenuates airway inflammation and hyper-responsiveness, prevents vascular leakage, and may be used as a therapeutic agent in allergic airway disease.

The present study examined the effects of cartilage oligometric matrix protein angiopoietin-1 (COMP-Ang1) on the revascularization of mice skin grafts. Full-thickness skin grafts were autotransferred into BALB/c mice. The donor grafts were soaked in COMP-Ang1 protein (50 μg/ml, n= 10) or in bovine serum albumin (BSA) (50 μg/ml, n= 10) dissolved in 1 ml of sterile, phosphate-buffered saline for 5 minutes before transfer. Revascularization of the grafts was monitored using an intravital microscope on postoperative days 3, 4, and 5. Morphological and immunohistochemical analyses were performed to evaluate platelet-endothelial cell adhesion molecule-1 and survivin expression and apoptotic signal in the transplanted grafts. Grafts soaked in COMP-Ang1 (COMP-Ang1 group) showed significantly increased revascularization compared with grafts soaked in BSA (BSA group) on intravital microscopy and platelet-endothelial cell adhesion molecule-1 staining. The COMP-Ang1 group showed a significant increase of survivin expression in the endothelial cells and a reduction of apoptotic signal in comparison to the BSA group. Therefore, we believe that COMP-Ang1 provides the therapeutic benefit of enhancing the survival of vascular endothelial cells during transplantation of skin graft.

Angiopoietin (Ang) signaling plays a role in angiogenesis and remodeling of blood vessels through the receptor tyrosine kinase Tie2, which is expressed on blood vessel endothelial cells (BECs). Recently it has been shown that Ang-2 is crucial for the formation of lymphatic vasculature and that defects in lymphangiogenesis seen in Ang-2 mutant mice are rescued by Ang-1. These findings suggest important roles for Ang signaling in the lymphatic vessel system; however, Ang function in lymphangiogenesis has not been characterized. In this study, we reveal that lymphatic vascular endothelial hyaluronan receptor 1-positive (LYVE-1+) lymphatic endothelial cells (LECs) express Tie2 in both embryonic and adult settings, indicating that Ang signaling occurs in lymphatic vessels. Therefore, we examined whether Ang-1 acts on in vivo lymphatic angiogenesis and in vitro growth of LECs. A chimeric form of Ang-1, cartilage oligomeric matrix protein (COMP)-Ang-1, promotes in vivo lymphatic angiogenesis in mouse cornea. Moreover, we found that COMP-Ang-1 stimulates in vitro colony formation of LECs. These Ang-1-induced in vivo and in vitro effects on LECs were suppressed by soluble Tie2-Fc fusion protein, which acts as an inhibitor by sequestering Ang-1. On the basis of these observations, we propose that Ang signaling regulates lymphatic vessel formation through Tie2. (Blood. 2005;105:4649-4656)

Fractalkine is an unusual tumor necrosis factor (TNF)-α-induced chemokine. The molecule is tethered to cells that express it and produces strong and direct adhesion to leukocytes expressing fractalkine receptor. However, the potential mechanism and significance of TNF-α-induced fractalkine expression in vascular endothelial cells are poorly understood. Here we show that in primary cultured endothelial cells TNF-α-induced fractalkine mRNA expression is mediated mainly through phosphatidylinositol 3′-kinase activation and nuclear factor (NF)-κB mediated transcriptional activation, along with GC-rich DNA-binding protein-mediated transcription. Interestingly, GC-rich DNA-binding protein inhibitors, mithramycin A and chromomycin A3, strongly suppressed TNF-α-induced fractalkine mRNA expression, possibly through inhibition of transcriptional activities by NF-κB and Sp1. In fact, direct inhibition of NF-κB and Sp1 bindings by decoy oligonucleotides suppressed TNF-α-induced fractalkine expression. Histologically, TNF-α-induced fractalkine expression was observed markedly in arterial and capillary endothelial cells, endocardium, and endothelium of intestinal villi, and slightly in venous endothelial cells, but not at all in lymphatic endothelial cells of intestine. Mithramycin A markedly suppressed TNF-α-induced fractalkine expression in vivo. These results indicate that TNF-α-stimulated fractalkine expression could act as part of arterial endothelial adhesion to leukocytes and monocytes during inflammation and atherosclerosis. NF-κB and Sp1 inhibitors such as mithramycin A may provide a pharmacological approach to suppressing these processes.

The mechanical overload of the heart is known to induce the expression of atrial natriuretic peptide (ANP) and heat-shock protein 70 (HSP70) in the heart. However, the relationship between these two substances remains unknown. In the present study, we characterized ANP secretion from mouse atria and examined a possible role that HSP70 may play in the regulation of ANP synthesis and secretion by using atria in transgenic mice in which HSP70 was overexpressed. We generated transgenic mice harboring the human HSP70 gene under the transcriptional control of human myosin heavy-chain promoter. In these mice, the transgene was overexpressed in the heart. Both atrial ANP messenger RNA and its concentration in the HSP70 transgenic mice were measured; these were not significantly different from those in wild-type mice. In isolated perfused non-beating atria, basal secretion of ANP was similar in both groups. When atrial volume was increased by changing atrial pressure, extracellular fluid (ECF) translocation and ANP secretion proportionately increased. Changes in atrial volume and ECF translocation and ANP secretion were positively correlated. However, these parameters did not significantly differ between the two groups. Endothelin-1 (ET-1), the strongest paracrine stimulus of ANP secretion, accentuated stretch-activated ANP secretion without significantly changing mechanically stimulated ECF translocation, as compared with that in the wild-type mice. The increased ANP secretion due to ET-1 in the transgenic mice was similar to that in the wild-type mice. The results suggest that both atrial stretching and ET-1 are important stimuli to ANP secretion from mouse atria, and the responsiveness of the ANP system to those stimuli are unlikely coupled to the pathway involving HSP70.

We defined whether extracorporeal shock wave therapy (ESWT) to the kidney activates the nitric oxide (NO)-cyclic 3′,5′-guanosine monophosphate (cGMP) pathway.

Mammalian aminoacyl tRNA synthetases form a macromolecular protein complex with three non-enzymatic cofactors. Among these factors, p43 is also secreted to work as a cytokine on endothelial as well as immune cells. Here we investigated the activity of p43 in angiogenesis and determined the related mediators. It promoted the migration of endothelial cells at low dose but induced their apoptosis at high dose. p43 at low concentration activated extracellular signal-regulating kinase, which resulted in the induction and activation of matrix metalloproteinase 9. In contrast, p43 at high concentration activated Jun N-terminal kinase, which mediated apoptosis of endothelial cells. These results suggest that p43 is a novel cytokine playing a dose-dependent biphasic role in angiogenesis.

A variety of surface receptors eliciting diverse cellular responses have been shown to recruit tumor necrosis factor receptor-associated factor (TRAF) adaptor molecules. However, a few TRAF-interacting intracellular proteins that serve as downstream targets or regulators of TRAF function have been identified. In search of new intracellular molecules that bind TRAF6, we carried out a yeast two-hybrid cDNA library screening with an N-terminal segment of TRAF6 as the bait. A novel human C2H2-type zinc finger family protein was identified, which when coexpressed with TRAF6 led to a suppression of TRAF6-induced activation of NF-κB and c-Jun N-terminal kinase. This novel protein was designated TIZ (forTRAF6-inhibitory zinc finger protein). TIZ expression also inhibited the signaling of RANK (receptor activator ofNF-κB), which together with TRAF6 has been shown to be essential for osteoclastogenesis. Furthermore, the expression level of TIZ appeared to be regulated during the differentiation of human peripheral blood monocytes into osteoclasts. More significantly, transfection of TIZ into the monocyte/macrophage cell line Raw264.7 reduced the RANK ligand-induced osteoclastogenesis of this cell line. Our findings suggest that the novel zinc finger protein TIZ may play a role during osteoclast differentiation by modulating TRAF6 signaling activity.

Until recently, vascular endothelial growth factor (VEGF) was the only growth factor proven to be specific and critical for blood vessel formation. Other long-known factors, such as the fibroblast growth factors (FGFs), platelet-derived growth factor, or transforming growth factor-ß, had profound effects in endothelial cells. But such factors were nonspecific, in that they could act on many other cells, and it seemed unlikely that these growth factors would be effective targets for treatment of endothelial cell diseases. A recently discovered endothelial cell specific growth factor, angiopoietin, has greatly contributed to our understanding of the development, physiology, and pathology of endothelial cells (Davis et al., 1996; Yancopoulos et al., 2000). The recent studies that identified and characterized the physiological and pathological roles of angiopoietin have allowed us to widen and deepen our knowledge about blood vessel formation and vascular endothelial function. Therefore, in this review, we describe the biomedical significance of these endothelial cell growth factors, the angiopoietins, in the vascular system under normal and pathological states.

The concept of “selective insulin resistance” has emerged as a unifying hypothesis in attempts to reconcile the influence of insulin resistance with that of hyperinsulinemia in the pathogenesis of macrovascular complications of diabetes. To explore this hypothesis in endothelial cells, we designed a set of experiments to mimic the “typical metabolic insulin resistance” by blocking the phosphatidylinositol 3-kinase pathway and exposing the cells to increasing concentrations of insulin (“compensatory hyperinsulinemia”). Inhibition of phosphatidylinositol 3-kinase with wortmannin blocked the ability of insulin to stimulate increased expression of endothelial nitric-oxide synthase, did not affect insulin-induced activation of MAP kinase, and increased the effects of insulin on prenylation of Ras and Rho proteins. At the same time, this experimental paradigm resulted in increased expression of vascular cellular adhesion molecules-1 and E-selectin, as well as increased rolling interactions of monocytes with endothelial cells. We conclude that inhibition of the metabolic branch of insulin signaling leads to an enhanced mitogenic action of insulin in endothelial cells.

Normally, tissue factor (TF) is not expressed on the surface of endothelial cells, but its expression can be induced by vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)‐α. However, the signaling pathway(s) affecting this induction is unknown. Using human umbilical vein endothelial cells, we found that inhibitors of guanine‐cytosine‐rich DNA binding protein and nuclear factor (NF)‐κB suppressed VEGF‐ and TNF‐α‐induced expression and activity of TF. However, unexpectedly, phosphatidylinositol (PI) 3′‐kinase inhibitor enhanced the VEGF‐ and TNF‐α‐induced expression and activity of TF. Angiopoietin‐1 (Ang1), a strong activator of intracellular PI 3′‐kinase/Akt, inhibited the induction of TF by VEGF and TNF‐α, whereas Ang1 itself did not produce any significant effect on TF. Selective activation (or inactivation) of PI 3′‐kinase/Akt by using adenoviral transfer reduced (or enhanced) TNF‐α‐induced expression of TF mRNA and protein, regardless of Ang1 treatment. From these results, we conclude that Ang1 inhibits the up‐regulation of TF expression, possibly through activation of PI 3′‐kinase/Akt in endothelial cells. Ang1 may be useful as an inhibitor of VEGF‐ and TNF‐α‐induced coagulation, inflammation, and cancer progression.

Background:Toluene diisocyanate (TDI) is a leading cause of occupational asthma. TDI-induced asthma is an inflammatory disease of the airways that is associated with airway remodeling. However, there are little data available on the role of matrix metalloproteinases (MMPs) in TDI-induced asthma. Objective:We evaluated whether MMP-9 participates in the airway inflammation in TDI-induced asthma. An additional aim of the present study was to determine whether MMP inhibitors could be effective therapeutic agents for TDI-induced asthma. Methods:We developed a murine model of TDI-induced asthma to examine the involvement of MMPs by performing 2 sensitizations with 3% TDI and 1 challenge with 1% TDI using ultrasonic nebulization. Results:Murine TDI-induced asthma includes findings of (1) increased inflammatory cells, including neutrophils, lymphocytes, and eosinophils; (2) histologic changes, including infiltration of inflammatory cells around bronchioles, thickened airway epithelium, and accumulation of mucus and debris in the bronchioles; (3) increased MMP-9 activity in inflammatory cells in the airway lumen; and (4) airway hyperresponsiveness. Administration of an MMP inhibitor remarkably reduced all these pathophysiologic findings. Conclusion:We conclude that TDI-induced occupational asthma is associated with the induction of MMP-9 in inflammatory cells, and the inhibition of MMP-9 may be a good therapeutic strategy. (J Allergy Clin Immunol 2001;108:1021-6.)

Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2443(Ang2443), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96–148) is absent in Ang2443because of alternative splicing of the gene. Like Ang2, recombinant Ang2443expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2443binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2443on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression ofAng2443mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2443mRNA and protein. Macrophages express mainly Ang2mRNA, but the expression of Ang2443mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2443is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.

The angiopoietin-Tie2 system is an important regulator of vasculogenesis and vascular integrity. Angiopoietin-2 (Ang2) disrupts blood vessel formation in the developing embryo by antagonizing the effects of angiopoietin-1 (Ang1) on the Tie2 receptor. In this study, we examined the effect of a well-known proinflammatory cytokine, tumor necrosis factor-α (TNF-α), on Ang2 expression in human umbilical vein endothelial cells. Reverse transcriptase-polymerase chain reaction and Northern blot analyses indicated that TNF-α induced Ang2 mRNA expression in a time- and dose-dependent manner. Western blot analyses revealed that TNF-α treatment increased cellular Ang2 protein. TNF-α induced less Ang2 mRNA expression in the presence of nuclear factor-κB (NF-κB) inhibitor. These results suggest that TNF-α-induced inflammatory angiogenesis might be facilitated by the induction of Ang2.

Using degenerate PCR we isolated a cDNA encoding a novel 406- and 410-amino acid protein from human and mouse embryonic cDNAs and have designated it ‘hepatic fibrinogen/angiopoietin-related protein’ (HFARP). The N-terminal and C-terminal portions of HFARP contain the characteristic coiled-coil domains and fibrinogen-like domains that are conserved in angiopoietins. In human and mouse tissues, HFARP mRNA is specifically expressed in the liver. HFARP mRNA and protein are mainly present in the hepatocytes. HFARP has a highly hydrophobic region at the N-terminus that is typical of a secretory signal sequence and one consensus glycosylation site. Recombinant HFARP expressed in COS-7 cells is secreted and glycosylated. HFARP protein is present not only in the hepatocytes, but also in the circulating blood. Recombinant HFARP acts as an apoptosis survival factor for vascular endothelial cells, but does not bind to Tie1 or Tie2 (endothelial-cell tyrosine kinase receptors). These results suggest that HFARP may exert a protective function on endothelial cells through an endocrine action.

The present study was designed to determine the spatial correlation among extent of DNA synthetic activity, expressions of G1/S phase cyclins, cyclin-dependent kinases (CDKs) and CIP/KIP family of CDK inhibitors (CKIs), and activities of G1/S phase CDKs in glomeruli and outer medullae of kidneys during the active regeneration period after ischemic injury. DNA synthetic activity was measured using [3H]-thymidine autoradiogram in the kidney sections. Cyclin, CDK, and CKI proteins were determined by Western blot analysis. CDK activities were determined by phosphorylation amount using specific substrate. The protein levels of cyclins (D1, D3, E, A) and activities of CDK4 and CDK2 were increased concomitant with the induction of DNA synthetic activity in outer medullae, but not in glomeruli, in adult kidneys during DNA synthetic period after ischemic injury. The p27KIP1 protein, but not the p21CIP1 protein, increased equally in total kidney, glomeruli, and outer medullae after ischemic injury. These results indicate that renal tubules have an active cyclin/CDK system, while glomeruli, do not have a cyclin/CDK system during active regeneration of kidneys after ischemic injury.

Using degenerate polymerase chain reaction, we isolated a cDNA encoding a novel 493-amino acid protein from human and mouse adult heart cDNAs and have designated it angiopoietin-related protein-2 (ARP2). The NH2-terminal and COOH-terminal portions of ARP2 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in angiopoietins. ARP2 has two consensus glycosylation sites and a highly hydrophobic region at the NH2terminus that is typical of a secretory signal sequence. Recombinant ARP2 expressed in COS cells is secreted and glycosylated. In human adult tissues, ARP2mRNA is most abundant in heart, small intestine, spleen, and stomach. In rat embryos, ARP2mRNA is most abundant in the blood vessels and skeletal muscles. Endothelial and vascular smooth muscle cells also contain ARP2mRNA. Recombinant ARP2 protein induces sprouting in vascular endothelial cells but does not bind to the Tie1 or Tie2 receptor. These results suggest that ARP2 may exert a function on endothelial cells through autocrine or paracrine action.

We examined the effect of angiopoietin‐1 (Ang1) on apoptosis in human umbilical vein endothelial cells (HUVECs). Ang1 (5–1000 ng/ml) dose‐dependently inhibited apoptosis under a serum‐deprived state. A significant apoptotic inhibition occurred with as low as 50 ng/ml. Two hundred ng/ml of Ang1 inhibited to approximately 50% of the control apoptotic rates for 96 h. Furthermore, an augmented antiapoptotic effect of Ang1 by the addition of 20 ng/ml vascular endothelial growth factor was observed. This Ang1‐induced strong antiapoptotic effect in endothelial cells is a novel and intriguing finding and could be an additional description of Ang1‐induced direct biological function.

Alternatively spliced isoforms of Na+–Ca2+exchanger were found from various tissues and species. RT-PCR amplification was performed on the basis of our cloned mouse cardiac Na+–Ca2+exchanger and four alternatively spliced isoforms of Na+–Ca2+exchanger were identified. Three (NCX1.3, NCX1.4, and NCX1.12) of them were first identified in the heart, and one isoform (NCX1.12) was a novel spliced variant. These four spliced variants were present in the embryonic and adult atria and ventricles. Different cell types of the heart expressed different spliced isoforms of Na+–Ca2+exchanger. Southern blot analysis indicated that the Na+–Ca2+exchanger gene existed as a single copy in the mouse genome. Thus, the Na+–Ca2+exchanger isoforms expressed in mouse heart are consistent with being produced by alternative splicing and they may have different functions in various cell types in the mouse heart.

The molecular mechanisms that arrest cardiomyocytes in the cell cycle during the postnatal period remain largely unknown. We have examined changes of the expression of cyclins and CDKs, the activity of each CDK in cardiomyocytes during the postnatal period, and have compared those changes with rate of binucleation formation of cardiomyocytes in rats. The mRNA and protein levels of cyclin D1, A and B in cardiomyocytes were high at day 1, then the levels decreased at different rates during the postnatal period. While the protein levels of cyclin A and B rapidly decreased, the protein level of cyclin D1 was relatively constant. The protein levels of CDK4, CDK2, and cdc2 in cardiomyocytes were high at day 1, then their levels gradually decreased. However, the activity of CDK4, which is responsible for G1 phase of cell cycle, was detectable only at day 1. The activity of CDK2 activity, which is responsible for the S phase of cell cycle, was relatively high at day 1, decreased at day 2, abruptly decreased at day 4, maintained the same low level until day 10, and barely or not detectable thereafter in cardiomyocytes. The activity of cdc2 was high at day 1, increased by 20% at day 2, and then gradually decreased thereafter, although approximately 50% of maximum activity was present at day 6. Most cardiomyocytes were mononucleated during the first 2 days postnatal. The percentage of binucleated cardiomyocytes increased from 2.5% at day 2, 14% at day 4, 50% at day 8, 80% at day 14, and had reached adult levels at day 21 after birth. During active binucleation formation in neonatal (from days 1–14) cardiomyocytes, CDK4 or CDK2 was functionally negligible, while cdc2 was functionally active. These data suggest that there were differential and dramatic decrease of CDK4 and CDK2 activities in cardiomyocytes during neonatal period, and the functionally active cdc2 in neonatal cardiomyocytes may be involved in binucleation formation.

Using a newly developed polyclonal antibody against murine cyclin D3, we have found that protein levels of cyclin D3 were highly detectable only in thymus and testis in rats. Since testis offer unique opportunities to examine the cell cyclein vivo,we examined the temporal and spatial expression of cyclin D3 and the DNA synthesis indicator, proliferating cell nuclear antigen (PCNA), in the rat testis during development. The protein levels of cyclin D3 protein in testis from 7 days to 3 months old were almost constant and then decreased gradually thereafter. The protein levels of cyclin D1 and PCNA were high in the testis of 7- and 14-day-old rats and decreased during testicular development. In the seminiferous tubules of 7-day-old newborns, cyclin D3 was surprisingly located in cytoplasm of stem cells that had bigger nuclei than the nuclei of surrounding cells. Interestingly, cyclin D3 immunopositive cells did not immunostain with PCNA in nuclei. In the adult testis, anti-cyclin D3 antibody strongly stained the cytoplasm of early stage primary spermatocytes, lightly stained pachytene spermatocytes, but did not stain elongated spermatids. There was no detectable cyclin D3 in Sertoli cells, interstitial cells, or fibroblasts within seminiferous tubules, or in blood vessels within the interstitial matrix. The known cyclin D3 partner, cyclin dependent kinase 4, was located mainly in nuclei of spermatogonia and in early stage primary spermatocytes. Strong PCNA immunopositive staining was located in the nuclei of spermatogonia in adult testis. These results indicate that cyclin D3 is detectable in meiotically active male germ cells (PCNA-negative cells), but is conspicuously absent from mitotically active spermatogonia (PCNA-positive cells). Moreover, in contrast toin vitroreports, cyclin D3 is not located in the nucleus, but rather in the cytoplasm of male germ cellsin vivo.Taken together, the presence of cyclin D3 in spermatocytes and its location in the cytoplasm lead us to speculate that cyclin D3 may have functions in male germ cells other than mitosis.

We have previously shown that there were differential and dramatic decreases of cyclin and cyclin-dependent kinase (CDK) activities in cardiomyocytes during the neonatal period. The activity of CDKs control cell cycle progression, and this activity is regulated positively and negatively by association of CDKs with cyclins and cyclin-dependent kinase inhibitors (CKIs), respectively. While the INK family (p15INK4B/p16INK4A/p18INK4C/p19INK4D) of CKIs is not detectable in hearts, the KIP/CIP family (p21CIP1, p27KIP1and p57KIP2) of CKIs is detectable in most organs including the heart. Differential and dramatic changes of the KIP/CIP family (p21CIP1, p27KIP1and p57KIP2) of CKIs were detected in rat hearts during development. The mRNA and protein levels of p21CIP1and p57KIP2were readily detectable in hearts at gestational and early postnatal periods and decreased thereafter. The mRNA levels of p27KIP1in ventricles were high during the gestational period, and did not change until day 30 postnatal, then were decreased slightly in 90-day-old rats. The protein levels of p27KIP1increased significantly in the early postnatal period, then were expressed persistently, although levels decreased slightly in the adult period. However, protein levels of p27KIP1in atria did not change during development. Variable immuno-staining patterns of p27KIP1were observed at different periods of development and in various locations in myocardium. During the gestational period, approximately 35–50% of myocardial cells in the cardiac wall were p27KIP1immuno-positive and were distributed diffusely. These p27KIP1immunopositive cells increased predominantly in endocardial and mid-portion areas of ventricular myocardium at the early postnatal period. This heterogenous pattern of p27KIP1protein expression persisted to adult hearts though the percentage of p27KIP1immuno-positive cells decreased slightly. High magnification revealed that more than 50% of adult cardiomyocytes were p27KIP1immuno-positive and that p27KIP1was located solely in nuclei. These results indicate that p27KIP1may be an important inhibitor of CDK activities in cardiomyocytes during early postnatal development and may block the re-entrance of adult cardiomyocytes into the cell cycle after injury.