Lymphatic vessels develop from specialized endothelial cells in preexisting blood vessels, but the molecular signals that regulate this separation are unknown. Here we identify a failure to separate emerging lymphatic vessels from blood vessels in mice lacking the hematopoietic signaling protein SLP-76 or Syk. Blood-lymphatic connections lead to embryonic hemorrhage and arteriovenous shunting. Expression of slp-76 could not be detected in endothelial cells, and blood-filled lymphatics also arose in wild-type mice reconstituted with SLP-76 -deficient bone marrow. These studies reveal a hematopoietic signaling pathway required for separation of the two major vascular networks in mammals.Mammals have two circulatory systems, a closed blood vasculature and an open lymphatic vasculature, that operate in parallel but develop in series (1,2). Although derived from venous endothelial precursors, lymphatic vessels do not communicate with blood vessels except at a single point where the thoracic duct empties into the subclavian vein (1-3). Recent studies have identified specific transcription factors and growth factors required to regulate the development † To whom correspondence should be addressed.
In contrast to lower vertebrates, the mammalian heart has limited capacity to regenerate after injury in part due to ineffective reactivation of cardiomyocyte proliferation. We show that the microRNA cluster miR302–367 is important for cardiomyocyte proliferation during development and is sufficient to induce cardiomyocyte proliferation in the adult and promote cardiac regeneration. In mice, loss of miR302–367 led to decreased cardiomyocyte proliferation during development. In contrast, increased miR302–367 expression led to a profound increase in cardiomyocyte proliferation, in part through repression of the Hippo signal transduction pathway. Postnatal reexpression of miR302–367 reactivated the cell cycle in cardiomyocytes, resulting in reduced scar formation after experimental myocardial infarction. However, long-term expression of miR302–367 induced cardiomyocyte dedifferentiation and dysfunction, suggesting that persistent reactivation of the cell cycle in postnatal cardiomyocytes is not desirable. This limitation can be overcome by transient systemic application of miR302–367 mimics, leading to increased cardiomyocyte proliferation and mass, decreased fibrosis, and improved function after injury. Our data demonstrate the ability of microRNA-based therapeutic approaches to promote mammalian cardiac repair and regeneration through the transient activation of cardiomyocyte proliferation.
Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function, KRIT1, CCM2 and PDCD10. Here we show that the HEG1 receptor, linked to CCM genes in zebrafish, is selectively expressed in endothelial cells and that Heg1-/- mice exhibit defective integrity of the heart, blood vessels and lymphatic vessels. In contrast, Heg1-/-;Ccm2+/lacZ and Ccm2lacZ/lacZ mice die early in development due to a failure of nascent endothelial cells to associate into patent vessels, a phenotype shared by deficient zebrafish embryos and reproduced by deficient endothelial cells ex vivo. These cardiovascular defects are associated with abnormal endothelial junctions like those observed in human CCMs, and biochemical and cellular imaging studies identify a cell autonomous pathway in which HEG1 receptors couple to KRIT1 at cell junctions. These studies identify HEG1-CCM signaling as a critical regulator of cardiovascular organ formation and integrity.
Mammals transport blood through a high-pressure, closed vascular network and lymph through a lowpressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life.
MicroRNA-based therapies that target cardiomyocyte proliferation have great potential for the treatment of myocardial infarction (MI). In previous work, we showed that the miR-302/367 cluster regulates cardiomyocyte proliferation in the prenatal and postnatal heart. Here, we describe the development and application of an injectable hyaluronic acid (HA) hydrogel for the local and sustained delivery of miR-302 mimics to the heart. We show that the miR-302 mimics released in vitro promoted cardiomyocyte proliferation over one week, and that a single injection of the hydrogel in the mouse heart led to local and sustained cardiomyocyte proliferation for two weeks. After MI, gel/miR-302 injection caused local clonal proliferation and increased cardiomyocyte numbers in the border zone of a Confetti mouse model. Gel/miR-302 further decreased cardiac end-diastolic (39%) and end-systolic (50%) volumes, and improved ejection fraction (32%) and fractional shortening (64%) four weeks after MI and injection, compared to controls. Our findings suggest that biomaterial-based miRNA delivery systems can lead to improved outcomes in cardiac regeneration.
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