Newborn Endothelial Cells Research Articles

Cerebral angiogenesis ameliorates pathological disorders in Nemo-deficient mice with small-vessel disease

Cerebral small-vessel diseases (SVDs) often follow a progressive course. Little is known about the function of angiogenesis, which potentially induces regression of SVDs. Here, we investigated angiogenesis in a mouse model of incontinentia pigmenti (IP), a genetic disease comprising features of SVD. IP is caused by inactivating mutations of Nemo, the essential component of NF-κB signaling. When deleting Nemo in the majority of brain endothelial cells (NemobeKO mice), the transcriptional profile of vessels indicated cell proliferation. Brain endothelial cells expressed Ki67 and showed signs of DNA synthesis. In addition to cell proliferation, we observed sprouting and intussusceptive angiogenesis in NemobeKO mice. Angiogenesis occurred in all segments of the vasculature and in proximity to vessel rarefaction and tissue hypoxia. Apparently, NEMO was required for productive angiogenesis because endothelial cells that had escaped Nemo inactivation showed a higher proliferation rate than Nemo-deficient cells. Therefore, newborn endothelial cells were particularly vulnerable to ongoing recombination. When we interfered with productive angiogenesis by inducing ongoing ablation of Nemo, mice did not recover from IP manifestations but rather showed severe functional deficits. In summary, the data demonstrate that angiogenesis is present in this model of SVD and suggest that it may counterbalance the loss of vessels.

Lactate potentiates angiogenesis and neurogenesis in experimental intracerebral hemorrhage

Lactate accumulation has been observed in the brain with intracerebral hemorrhage (ICH). However, the outcome of lactate accumulation has not been well characterized. Here, we report that lactate accumulation contributes to angiogenesis and neurogenesis in ICH. In the first set of the experiment, a rat model of ICH was induced by injecting collagenase into the brain. The effects of lactate accumulation on the neurological function, apoptosis, and numbers of newborn endothelial cells and neurons, as well as the proliferation-associated signaling pathway, were evaluated in the rat brain. In the second set, exogenous l-lactate was infused into intact rat brains so that its effects could be further assessed. Following ICH, lactate accumulated around the hematoma; the numbers of PCNA+/vWF+ nuclei and PCNA+/DCX+ cells were significantly increased compared with the numbers in the Sham group. Moreover, ICH induced translocation of nuclear factor-kappa B (NF-κB) p65 into the nucleus, resulting in a notable upregulation of VEGF and bFGF mRNAs and proteins compared with the levels in the Sham controls. Administration of a lactate dehydrogenase inhibitor dramatically inhibited these effects, decreased the vascular density, and aggravated neurological severity scores and apoptosis after ICH. After exogenous l-lactate infusion, the numbers of PCNA+/vWF+ nuclei and PCNA+/DCX+ cells were strikingly increased compared with the numbers in the Sham controls. In addition, lactate facilitated NF-κB translocation to induce increased transcription of VEGF and bFGF. Co-infusion with an NF-κB inhibitor significantly inhibited these effects. These data suggest that lactate potentiates angiogenesis and neurogenesis by activating the NF-κB signaling pathway following ICH.

Systemic administration of cell-free exosomes generated by human bone marrow derived mesenchymal stem cells cultured under 2D and 3D conditions improves functional recovery in rats after traumatic brain injury

Multipotent human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after experimental traumatic brain injury (TBI). The present study was designed to investigate whether systemic administration of cell-free exosomes generated from hMSCs cultured in 2-dimensional (2D) conventional conditions or in 3-dimensional (3D) collagen scaffolds promote functional recovery and neurovascular remodeling in rats after TBI. Wistar rats were subjected to TBI induced by controlled cortical impact; 24 h later tail vein injection of exosomes derived from hMSCs cultured under 2D or 3D conditions or an equal number of liposomes as a treatment control were performed. The modified Morris water maze, neurological severity score and footfault tests were employed to evaluate cognitive and sensorimotor functional recovery. Animals were sacrificed at 35 days after TBI. Histological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. Compared with liposome-treated control, exosome-treatments did not reduce lesion size but significantly improved spatial learning at 33–35 days measured by the Morris water maze test, and sensorimotor functional recovery, i.e., reduced neurological deficits and footfault frequency, observed at 14–35 days post injury (p < 0.05). Exosome treatments significantly increased the number of newborn endothelial cells in the lesion boundary zone and dentate gyrus, and significantly increased the number of newborn mature neurons in the dentate gyrus as well as reduced neuroinflammation. Exosomes derived from hMSCs cultured in 3D scaffolds provided better outcome in spatial learning than exosomes from hMSCs cultured in the 2D condition. In conclusion, hMSC-generated exosomes significantly improve functional recovery in rats after TBI, at least in part, by promoting endogenous angiogenesis and neurogenesis and reducing neuroinflammation. Thus, exosomes derived from hMSCs may be a novel cell-free therapy for TBI, and hMSC-scaffold generated exosomes may selectively enhance spatial learning.

Immature endothelial cells initiate endothelin-mediated constriction of newborn arteries.

Endothelial expression and the release of endothelin-1 (ET-1) in levels sufficient to initiate vasoconstriction is considered to be a hallmark feature of pathological endothelial dysfunction. During the immediate postnatal period, arterial endothelial cells undergo remarkable structural and functional changes as they transition to a mature protective cell layer, which includes a marked increase in NO dilator activity. The present study demonstrates that endothelial cells lining newborn central arteries express high levels of ET-1 peptides and, in response to endothelial stimulation, rapidly release ET-1 and initiate powerful ET-1-mediated constriction. This activity is lost as the endothelium matures in the postnatal period. Heightened activity of ET-1 in the neonatal endothelium might contribute to inappropriate responses of immature arteries to stress or injury. Indeed, the immature endothelium resembles dysfunctional endothelial cells, and retention or re-emergence of this phenotype may contribute to the development of vascular disease. Endothelial cells lining fetal and newborn arteries have an unusual phenotype, including reduced NO activity, prominent actin stress fibres and poorly developed cellular junctions. Experiments were performed to determine whether the immature endothelium of newborn arteries also expresses and releases endothelin-1 (ET-1) and initiates endothelium-dependent constriction. Carotid arteries were isolated from newborn (postnatal day 1; P1), postnatal day 7 (P7) and postnatal day 21 (P21) mice and assessed in a pressure myograph system. Endothelial stimulation with A23187 or thrombin caused constriction in P1 arteries, no significant change in diameter of P7 arteries, and dilatation in P21 arteries. In P1 arteries, constriction to thrombin or A23187 was inhibited by endothelial-denudation, by ET-1 receptor antagonists (BQ123 plus BQ788) or by inhibition of endothelin-converting enzyme (phosphoramidon or SM19712). ET-1 receptor antagonism did not affect responses to thrombin or A23187 in more mature arteries. Exogenous ET-1 caused similar concentration-dependent constrictions of P1, P7 and P21 arteries. Endothelial stimulation with thrombin rapidly increased the endothelial release of ET-1 from P1 but not P21 aortas. Endothelial expression of ET-1 peptides, as assessed by immunofluorescence analysis, was increased in P1 compared to P21 arteries. Therefore, newborn endothelial cells express high levels of ET-1 peptides, rapidly release ET-1 in response to endothelial stimulation, and initiate ET-1-mediated endothelium-dependent constriction. This activity is diminished as the endothelium matures in the immediate postnatal period. Heightened activity of ET-1 in neonatal endothelium probably reflects an early developmental role of the peptide, although this might contribute to inappropriate responses of immature arteries to stress or injury.

A novel method for endothelial cell isolation.

The present study aimed to develop a quick and efficient method for purification of newborn endothelial cells from tumor tissues. Fresh tissues were separated from C57BL/6 mice bearing tumors derived from mouse lung cancer Lewis cells, fully minced and divided into two parts. One part was subjected to collagenase type I digestion with a vortex to form a single-cell suspension, while another part was digested but without a vortex. Then, the CD105+ cells were isolated using anti-CD105 antibody-coated Dynabeads. The isolated CD105+ cells were grown in culture medium and examined for the surface expression of CD105 by a fluorescence-activated cell sorter (FACS). The uptake of acetylated LDL and the ability to maintain capillary tube-like structure formation in the CD105+ cells were also examined by Dil-Ac-LDL uptake assay and tube formation assay. The expression of tumor newborn endothelial cells (CD105+) was tested in Lewis xenografts by immunohistochemistry. The number of cells which were obtained by the digestion process with a vortex was 5.70±0.23x10(4) much higher than the number without a vortex (0.32±0.04x10(4)) (P<0.01). The purity of CD105+ cell digestion with a vortex was significantly higher than that without a vortex. Dil-Ac-LDL uptake assay and tube formation assay confirmed that the CD105+ cells digested with a vortex exhibited typical functions of endothelial cells. In conclusion, the CD105+ cells isolated by the new method had high purity and displayed features of vascular endothelial cells. The modified method provides CD105+ cells with superior conditions for mechanistic research on the development of vessel-based disease.

Blood–brain barrier breakdown and repair by Src after thrombin‐induced injury

Thrombin mediates the life-threatening cerebral edema that occurs after intracerebral hemorrhage. Therefore, we examined the mechanisms of thrombin-induced injury to the blood-brain barrier (BBB) and subsequent mechanisms of BBB repair. Intracerebroventricular injection of thrombin (20U) was used to model intraventricular hemorrhage in adult rats. Thrombin reduced brain microvascular endothelial cell (BMVEC) and perivascular astrocyte immunoreactivity-indicating either cell injury or death-and functionally disrupted the BBB as measured by increased water content and extravasation of sodium fluorescein and Evans blue dyes 24 hours later. Administration of nonspecific Src family kinase inhibitor (PP2) immediately after thrombin injections blocked brain edema and BBB disruption. At 7 to 14 days after thrombin injections, newborn endothelial cells and astrocytes were observed around cerebral vessels at the time when BBB permeability and cerebral water content resolved. Delayed administration of PP2 on days 2 through 6 after thrombin injections prevented resolution of the edema and abnormal BBB permeability. Thrombin, via its protease-activated receptors, is postulated to activate Src kinase phosphorylation of molecules that acutely injure the BBB and produce edema. Thus, acute administration of Src antagonists blocks edema. In contrast, Src blockade for 2 to 6 days after thrombin injections is postulated to prevent resolution of edema and abnormal BBB permeability in part because Src kinase proto-oncogene members stimulate proliferation of newborn BMVECs and perivascular astrocytes in the neurovascular niche that repair the damaged BBB. Thus, Src kinases not only mediate acute BBB injury but also mediate chronic BBB repair after thrombin-induced injury.

Physical activity promotes angio- and vasculogenesis in the post-ischemic brain

Clinical and experimental evidence supports the notion that regular physical activity up regulates endothelial nitric oxide synthase, improves endothelium-dependent vasorelaxation, and protects from vascular disease. Here, we demonstrate that continuous voluntary training on running wheels via up regulation of endothelial nitric oxide synthase improves neo-vascularization and long-term recovery following mild brain ischemia induced by 30 min filamentous occlusion of the left middle cerebral artery followed by reperfusion in the mouse. Experiments were performed under isofluorane anesthesia in 129/SV wild-type mice or chimeric mice that were transplanted with bone marrow from transgenic mice that express the lacZ gene under control of the endothelial promotor Tie2. In ischemic animals, physical activity conferred up regulation of endothelial nitric oxide synthase in the vasculature and of endothelial progenitor cells in the spleen and bone marrow as measured by FACS analysis and cell culture techniques. This was associated with higher numbers of circulating endothelial progenitor cells in the blood, enhanced neo-vascularization in a disc angiogenesis model, and increased engraftment of Tie2/lacZ positive bone marrow-derived cells in the ischemic brain. Six weeks after the insult, trained animals had higher numbers of newborn endothelial cells in the brain (as determined by bromodesoxyuridine/von Willebrand factor double immunostaining and confocal microscopy) and increased density of perfused vessels with apparent normal morphology (as determined by tiled-field mapping of Evans Blue perfused brains). This conferred sustained augmentation of absolute regional cerebral blood flow in the ischemic lesion in trained vs. sedentary mice at 6 weeks after the insult (as determined by 14-C iodoantipyrine autoradiography). In fact, exercised mice had smaller ischemic lesion sizes (determined on NeuN-immunostained coronal brain sections by computer assisted volumetry), improved functional scores (Bederson test, wire hanging) and better performance in the Morris water maze when compared to sedentary animals. Co-administration of the nitric oxide synthase inhibitor L-NAME in the drinking water not only inhibited up regulation of endothelial progenitor cells by physical activity but also completely abrogated the stroke-protective effects of exercise. In conclusion, voluntary physical activity enhances regeneration in the ischemic brain by endothelial nitric oxide-dependent mechanisms leading to improved neo-vascularization and increased cerebral blood flow.

Vascular niche for adult hippocampal neurogenesis.

The thin lamina between the hippocampal hilus and granule cell layer, or subgranule zone (SGZ), is an area of active proliferation within the adult hippocampus known to generate new neurons throughout adult life. Although the neuronal fate of many dividing cells is well documented, little information is available about the phenotypes of cells in S-phase or how the dividing cells might interact with neighboring cells in the process of neurogenesis. Here, we make the unexpected observation that dividing cells are found in dense clusters associated with the vasculature and roughly 37% of all dividing cells are immunoreactive for endothelial markers. Most of the newborn endothelial cells disappear over several weeks, suggesting that neurogenesis is intimately associated with a process of active vascular recruitment and subsequent remodeling. The present data provide the first evidence that adult neurogenesis occurs within an angiogenic niche. This environment may provide a novel interface where mesenchyme-derived cells and circulating factors influence plasticity in the adult central nervous system.