Endothelial Cell Turnover Research Articles

Dynamics of Endothelial Cell Generation and Turnover in Arteries During Homeostasis and Diseases.

Endothelial cell (EC) generation and turnover by self-proliferation contributes to vascular repair and regeneration. The ability to accurately measure the dynamics of EC generation would advance our understanding of cellular mechanisms of vascular homeostasis and diseases. However, it is currently challenging to evaluate the dynamics of EC generation in large vessels such as arteries because of their infrequent proliferation. By using dual recombination systems based on Cre-loxP and Dre-rox, we developed a genetic system for temporally seamless recording of EC proliferation in vivo. We combined genetic recording of EC proliferation with single-cell RNA sequencing and gene knockout to uncover cellular and molecular mechanisms underlying EC generation in arteries during homeostasis and disease. Genetic proliferation tracing reveals that ≈3% of aortic ECs undergo proliferation per month in adult mice during homeostasis. The orientation of aortic EC division is generally parallel to blood flow in the aorta, which is regulated by the mechanosensing protein Piezo1. Single-cell RNA sequencing analysis reveals 4 heterogeneous aortic EC subpopulations with distinct proliferative activity. EC cluster 1 exhibits transit-amplifying cell features with preferential proliferative capacity and enriched expression of stem cell markers such as Sca1 and Sox18. EC proliferation increases in hypertension but decreases in type 2 diabetes, coinciding with changes in the extent of EC cluster 1 proliferation. Combined gene knockout and proliferation tracing reveals that Hippo/vascular endothelial growth factor receptor 2 signaling pathways regulate EC proliferation in large vessels. Genetic proliferation tracing quantitatively delineates the dynamics of EC generation and turnover, as well as EC division orientation, in large vessels during homeostasis and disease. An EC subpopulation in the aorta exhibits more robust cell proliferation during homeostasis and type 2 diabetes, identifying it as a potential therapeutic target for vascular repair and regeneration.

The G3BP1-UPF1-Associated Long Non-Coding RNA CALA Regulates RNA Turnover in the Cytoplasm.

Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.

Sclerotherapy for Oral Vascular Malformation : case series

Vascular malformations are structural abnormalities in blood vessels with normal endothelial cell turnover, and are known as one of vascular abnormalities along with hemangiomas. Vascular malformations can occur anywhere in the body and are relatively rare in the oral cavity. It presents symptoms such as bleeding, pain, and swelling, and can cause functional and aesthetic problems. Treatment methods for vascular malformations include surgical excision, Nd:YAG and CO2 laser therapy, embolization and sclerotherapy. In this study, three cases of vascular malformations in the soft tissues of the oral cavity were identified. We report three cases who were simply treated using sclerotherapy with a review of the literature. Sclerotherapy using a sclerosant showed successful results, and known complications were not observed. In properly selected patients with vascular malformation, sclerotherapy can be considered as the first treatment.

Dysfunctional Vascular Endothelium as a Driver of Atherosclerosis: Emerging Insights Into Pathogenesis and Treatment.

Atherosclerosis, the chronic accumulation of cholesterol-rich plaque within arteries, is associated with a broad spectrum of cardiovascular diseases including myocardial infarction, aortic aneurysm, peripheral vascular disease, and stroke. Atherosclerotic cardiovascular disease remains a leading cause of mortality in high-income countries and recent years have witnessed a notable increase in prevalence within low- and middle-income regions of the world. Considering this prominent and evolving global burden, there is a need to identify the cellular mechanisms that underlie the pathogenesis of atherosclerosis to discover novel therapeutic targets for preventing or mitigating its clinical sequelae. Despite decades of research, we still do not fully understand the complex cell-cell interactions that drive atherosclerosis, but new investigative approaches are rapidly shedding light on these essential mechanisms. The vascular endothelium resides at the interface of systemic circulation and the underlying vessel wall and plays an essential role in governing pathophysiological processes during atherogenesis. In this review, we present emerging evidence that implicates the activated endothelium as a driver of atherosclerosis by directing site-specificity of plaque formation and by promoting plaque development through intracellular processes, which regulate endothelial cell proliferation and turnover, metabolism, permeability, and plasticity. Moreover, we highlight novel mechanisms of intercellular communication by which endothelial cells modulate the activity of key vascular cell populations involved in atherogenesis, and discuss how endothelial cells contribute to resolution biology – a process that is dysregulated in advanced plaques. Finally, we describe important future directions for preclinical atherosclerosis research, including epigenetic and targeted therapies, to limit the progression of atherosclerosis in at-risk or affected patients.

Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

Abstract After myocardial infarct (MI), followed by ischemia and scar formation, interstitial cells play key roles in the adaptation to injury. Endothelial cells (ECs), for instance, can clonally expand, migrate into the infarct area and facilitate crucial functions promoting revascularization, reestablishment of oxygen supply and secretion of paracrine factors. Moreover, ECs can transiently undergo changes towards a mesenchymal phenotype (Endothelial-to-mesenchymal transition; EndMT). Whether this process contributes to long-term cardiac fibrosis or helps to facilitate post-ischemic vessel growth remains controversial. Here, we aim to delineate kinetics and characteristics of phenotypic changes in ECs with single cell RNA-sequencing (scRNA-seq). We performed a time course (homeostasis or 0 day (d), 1d, 3d, 5d, 7d, 14d, 28d post-MI) in mice and isolated the non-cardiomyocyte fraction for scRNA-seq (n=35,312 cells). Pecam1/Cdh5 double positive ECs showed expression of apoptosis, hypoxia and inflammation markers at 3d. Bioinformatic cell cycle analysis predicted high association with proliferative capacities at 3d, indicative of EC turnover post-MI. Metabolism, recently linked to regulate EndMT, was altered. We found genes of the glycolysis and the TCA-cycle pathway upregulated at 1d to 3d, and a decrease of fatty acid signaling genes. At 3d, mesenchymal markers Fn1, Vim, S100a4, Serpine1 transiently increased compared to homeostasis (>1.6-fold, p<0.05) together with a reduction of EC genes such as Pecam1. Interestingly, mesenchymal transition was transient and returned to baseline levels at 28d after MI. Cell fate trajectory analysis confirmed these findings by identifying an EC state characterized by high proliferation and mesenchymal but low EC properties. At 3d to 7d the majority of the ECs were assigned to this state, based on their transcriptomic profile. We additionally used Cdh5-CreERT2; R26-mT/mG mice followed by scRNA-seq to trace the fate of ECs. Bioinformatic analysis of GFP-positive ECs confirmed the gain in mesenchymal marker but revealed no full transition to the mesenchymal state at later timepoints. This suggests a transient mesenchymal activation of ECs rather than a complete lineage transition. We further induced EndMT with TGF-β2 in ECs in vitro and observed reversibility of the phenotype after withdrawal of the stimulus. After treatment, ECs upregulated various mesenchymal marker genes. Withdrawal of TGF-β2 at 3d or 7d, reverted expression to baseline levels. We further determined DNA methylation of EndMT gene loci to assess if TGF-β2 leads to a true fate change but did not observe changes after TGF-β2 stimulation and withdrawal. Taken together, our data suggests that ECs undergo a transient mesenchymal activation concomitant with a metabolic adaptation early after MI but do not acquire a long-term mesenchymal fate. This activation may facilitate EC migration and clonal expansion to regenerate the vascular network. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Center of Cardiovascular Research (DZHK), Deutsche Forschungsgemeinschaft (DFG) CRC1366 Project B4

Case report of a large hemangioma of the parotid gland with multiple phleboliths and tonsilloliths

Hemangiomas are benign vascular deformities characterized by an increased proliferation and turnover of endothelial cells. They account for the majority of parotid gland tumors in infants but are rare in adults. Changes in blood flow dynamics within hemangiomas can cause stasis, thrombus formation and phleboliths. Tonsilloliths are calcifications occurring primarily within the palatine tonsillar crypts. We report the case of a large hemangioma of the parotid gland with multiple phleboliths and tonsilloliths in an adult, highlighting the clini cal and imaging features on panoramic radiography, Cone Beam Computed Tomography (CBCT), cervical ultrasound, Magnetic Resonance Imaging (MRI) and Doppler imaging. A 20-year-old woman presented, complaining of a painless swelling below her right ear. Panoramic radiograph showed multiple randomly distributed round-to-oval radiopaque structures overlying the right mandibular ramus. CBCT revealed several radiopaque structures in the right palatine tonsillar crypts. Cervical ultrasound exposed a large heterogeneous and predominantly hypoechogenic mass in the right parotid gland. MRI displayed a well-defined lesion in the right parotid gland extending into the parapharyngeal pre-styloid space, hypointense on T1 and hyperintense on T2, containing several nodules. The diagnosis was: large hemangioma of the right parotid gland extending into the parapharyngeal pre-styloid space, with multiple phleboliths and tonsilloliths. Propranolol was delivered, with periodic follow-up on Doppler images, showing a hemangioma size reduction. Standard radiographs can detect tonsilloliths and phleboliths but additional imaging modalities disclose the exact diagnosis and location of calcifications and the diagnosis, structure and extent of the vascular lesion. Tonsilloliths and phleboliths should be considered in the differential diagnosis of radiopaque masses involving the mandibular ramus. Hemangioma with phleboliths should be considered in the differential diagnosis of parotid tumors when numerous intraglandular calcification nodules are detected on radiographs.

Homeobox B9 integrates bone morphogenic protein 4 with inflammation at atheroprone sites.

AimsAtherosclerosis develops near branches and bends of arteries that are exposed to disturbed blood flow which exerts low wall shear stress (WSS). These mechanical conditions alter endothelial cells (EC) by priming them for inflammation and by inducing turnover. Homeobox (Hox) genes are developmental genes involved in the patterning of embryos along their anterior–posterior and proximal–distal axes. Here we identified Hox genes that are regulated by WSS and investigated their functions in adult arteries.Methods and resultsEC were isolated from inner (low WSS) and outer (high WSS) regions of the porcine aorta and the expression of Hox genes was analysed by quantitative real-time PCR. Several Hox genes (HoxA10, HoxB4, HoxB7, HoxB9, HoxD8, HoxD9) were significantly enriched at the low WSS compared to the high WSS region. Similarly, studies of cultured human umbilical vein EC (HUVEC) or porcine aortic EC revealed that the expression of multiple Hox genes (HoxA10, HoxB9, HoxD8, HoxD9) was enhanced under low (4 dyn/cm2) compared to high (13 dyn/cm2) WSS conditions. Gene silencing studies identified Hox genes (HoxB9, HoxD8, HoxD9) that are positive regulators of inflammatory molecule expression in EC exposed to low WSS, and others (HoxB9, HoxB7, HoxB4) that regulated EC turnover. We subsequently focused on HoxB9 because it was strongly up-regulated by low WSS and, uniquely, was a driver of both inflammation and proliferation. At a mechanistic level, we demonstrate using cultured EC and murine models that bone morphogenic protein 4 (BMP4) is an upstream regulator of HoxB9 which elicits inflammation via induction of numerous inflammatory mediators including TNF and downstream NF-κB activation. Moreover, the BMP4-HoxB9-TNF pathway was potentiated by hypercholesterolaemic conditions.ConclusionsLow WSS induces multiple Hox genes that control the activation state and turnover of EC. Notably, low WSS activates a BMP4-HoxB9-TNF signalling pathway to initiate focal arterial inflammation, thereby demonstrating integration of the BMP and Hox systems in vascular pathophysiology.

Cerebrovascular plasticity: Processes that lead to changes in the architecture of brain microvessels

The metabolic demands of the brain are met by oxygen and glucose, supplied by a complex hierarchical network of microvessels (arterioles, capillaries, and venules). Transient changes in neural activity are accommodated by local dilation of arterioles or capillaries to increase cerebral blood flow and hence nutrient availability. Transport and communication between the circulation and the brain is regulated by the brain microvascular endothelial cells that form the blood–brain barrier. Under homeostatic conditions, there is very little turnover in brain microvascular endothelial cells, and the cerebrovascular architecture is largely static. However, changes in the brain microenvironment, due to environmental factors, disease, or trauma, can result in additive or subtractive changes in cerebrovascular architecture. Additions occur by angiogenesis or vasculogenesis, whereas subtractions occur by vascular pruning, injury, or endothelial cell death. Here we review the various processes that lead to changes in the cerebrovascular architecture, including sustained changes in the brain microenvironment, development and aging, and injury, disease, and repair.

Endothelial Regenerative Capacity and Aging: Influence of Diet, Exercise and Obesity.

Background: The endothelium plays an important role in cardiovascular regulation, from blood flow to platelet aggregation, immune cell infiltration and demargination. A dysfunctional endo-thelium leads to the onset and progression of Cardiovascular Disease (CVD). The aging endothelium displays significant alterations in function, such as reduced vasomotor functions and reduced angio-genic capabilities. This could be partly due to elevated levels of oxidative stress and reduced endothe-lial cell turnover. Circulating angiogenic cells, such as Endothelial Progenitor Cells (EPCs) play a significant role in maintaining endothelial health and function, by supporting endothelial cell prolifera-tion, or via incorporation into the vasculature and differentiation into mature endothelial cells. Howev-er, these cells are reduced in number and function with age, which may contribute to the elevated CVD risk in this population. However, lifestyle factors, such as exercise, physical activity obesity, and dietary intake of omega-3 polyunsaturated fatty acids, nitrates, and antioxidants, significantly af-fect the number and function of these circulating angiogenic cells.Conclusion: This review will discuss the effects of advancing age on endothelial health and vascular regenerative capacity, as well as the influence of diet, exercise, and obesity on these cells, the mecha-nistic links and the subsequent impact on cardiovascular health

Does Multicolor Lineage Tracing of Endothelial Cells Provide a Black and White Answer on Clonal Expansion in Post-Natal Angiogenesis?

Postnatal angiogenesis occurs in physiological (ie, blood vessel growth in muscle in response to exercise and with cyclic changes within menstrual tissue) and pathological conditions (eg, diabetic retinopathy). Many attempts to promote postnatal angiogenesis have been attempted to drive revascularization of ischemic tissues. Surprisingly, we continue to lack fundamental information on which endothelial cells possess replicative potential in blood vessels. Prior work established that infusion of H3 thymidine into young mammals revealed endothelial cell proliferation in small and large vessels that is high at birth, varies within and among organ vasculature, and declines rapidly over the first few weeks of life. In adult animals, the rate of homeostatic endothelial cell turnover has been quantified as <1% in rodents. Despite these pieces of information, we do not know specifics about the individual proliferative capacities of endothelial cells, whether this activity is restricted to a small subset or is a feature ubiquitously present within all endothelial cells in a blood vessel.1 Article, see p 670 Manavski et al2 in this issue have used inducible endothelial cell–restricted multicolor reporter (Confetti) transgenic mice to interrogate the clonal expansion of individual endothelial cells within normally developing murine tissue vasculature or within tissues challenged by ischemia and hypoxia. This system has permitted quantification of all the endothelial cells within a fixed sampling volume of the tissue vasculature to detect an increase in local distribution of a singular color of endothelial cells within areas of clonal expansion compared with near equivalent levels of random color distribution in areas lacking clonal expansion. In brief, the authors failed to detect significant color enrichment (<10% clonal expansion) in the developing retinal vasculature at postnatal day 5, consistent with prior work in zebrafish that had identified random integration or cell mixing accounting for endothelial cell distributions in developmental …

Tumor angiogenesis. From bench to bedside

Under physiological conditions, angiogenesis is regulated by the local balance between endogenous stimulators and inhibitors. In recent years, evidence has accu- mulated that, in addition to the classic factors, many other endogenous peptides play an important regulatory role in angiogenesis, especially under pathological con- ditions. In chronic inflammation and tumor growth, there is an imbalance between endogenous stimulator and inhibitor levels, leading to an “angiogenic switch”. Vari - ous regulatory elements control the switch to the vascular phase. Endothelial cell turnover in the healthy adult organism is low, the quiescence being maintained by the dominant influence of endogenous angiogenesis inhibitors over angiogenic stim - uli. In pathological situations angiogenesis may be triggered not only by the over- production of pro-angiogenic factors, but also by the down-regulation of inhibitory factors. The stromal microenvironment is essential for cell proliferation and angiogen- esis through its provision of survival signals, secretion of growth and pro-angiogenic factors, and direct adhesion molecule interactions. Tumor cells are surrounded by an infiltrate of inflammatory cells, namely lymphocytes, neutrophils, macrophages, and mast cells, which communicate via a complex network of intercellular signaling path- ways mediated by surface adhesion molecules, cytokines, and their receptors. Much research effort has been concentrated on the role of angiogenesis in cancer, and inhi - bition of angiogenesis is a major area of therapeutic development for the treatment of this disease. New pathophysiological concepts generated in the past few decades have given rise to the development of a large variety of new drugs to interfere with angiogenesis. Angiogenesis inhibitors are now being approved and introduced into medical practice throughout the world and inhibition of angiogenesis is a major area of therapeutic development for the treatment of cancer. However, even if the major- ity of pre-clinical studies have shown that the growth of all experimental tumors can be effectively inhibited by various anti-angiogenic agents, the clinical benefits of anti- angiogenic treatments are relatively modest, and in the majority of cases, the drugs merely slow down tumor progression and prolong survival by only a few more months.

Experimental Branch Retinal Vein Occlusion Induces Upstream Pericyte Loss and Vascular Destabilization.

AimsBranch retinal vein occlusion (BRVO) leads to extensive vascular remodeling and is important cause of visual impairment. Although the vascular morphological changes following experimental vein occlusion have been described in a variety of models using angiography, the underlying cellular events are ill defined.Methods and ResultsWe here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC) apoptosis in the upstream vascular network associated with a transient edema and hemorrhages. Subsequently, we observe an induction of EC proliferation within the dilated vein and capillaries, detected by EdU incorporation, and the edema resolves. However, the pericytes of the upstream capillaries are severely reduced, which was associated with continuing EC apoptosis and proliferation. The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression. Exposure of the experimental animals to hypoxia, when pericyte (PC) dropout had occurred, led to a dramatic increase in endothelial cell proliferation, confirming the vascular instability induced by the experimental BRVO.ConclusionExperimental BRVO leads to acute endothelial cells apoptosis and increased permeability. Subsequently the upstream vascular network remains destabilized, characterized by pericyte dropout, un-physiologically high endothelial cells turnover and sensitivity to hypoxia. These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

Rap1 promotes endothelial mechanosensing complex formation, NO release and normal endothelial function.

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.

GATA4 promotes endothelial cell turnover at regions of arteries exposed to disturbed flow

Introduction: Blood flow generates wall shear stress (WSS) which influences endothelial cell (EC) physiology. Low, oscillatory WSS promotes atherosclerosis by increasing EC turnover and permeability to macromolecules. We previously used microarrays and computational fluid dynamics to study the EC transcriptome at mechanically-distinct regions of the porcine aorta, and demonstrated that the transcription factor GATA4 was enriched at sites exposed to low WSS. We hypothesised that GATA4 regulates turnover and permeability in EC monolayers exposed to disturbed flow.

Therapeutic potential of HDL in cardioprotection and tissue repair.

Epidemiological studies support a strong association between high-density lipoprotein (HDL) cholesterol levels and heart failure incidence. Experimental evidence from different angles supports the view that low HDL is unlikely an innocent bystander in the development of heart failure. HDL exerts direct cardioprotective effects, which are mediated via its interactions with the myocardium and more specifically with cardiomyocytes. HDL may improve cardiac function in several ways. Firstly, HDL may protect the heart against ischaemia/reperfusion injury resulting in a reduction of infarct size and thus in myocardial salvage. Secondly, HDL can improve cardiac function in the absence of ischaemic heart disease as illustrated by beneficial effects conferred by these lipoproteins in diabetic cardiomyopathy. Thirdly, HDL may improve cardiac function by reducing infarct expansion and by attenuating ventricular remodelling post-myocardial infarction. These different mechanisms are substantiated by in vitro, ex vivo, and in vivo intervention studies that applied treatment with native HDL, treatment with reconstituted HDL, or human apo A-I gene transfer. The effect of human apo A-I gene transfer on infarct expansion and ventricular remodelling post-myocardial infarction illustrates the beneficial effects of HDL on tissue repair. The role of HDL in tissue repair is further underpinned by the potent effects of these lipoproteins on endothelial progenitor cell number, function, and incorporation, which may in particular be relevant under conditions of high endothelial cell turnover. Furthermore, topical HDL therapy enhances cutaneous wound healing in different models. In conclusion, the development of HDL-targeted interventions in these strategically chosen therapeutic areas is supported by a strong clinical rationale and significant preclinical data.

Suppression of Colorectal Cancer Liver Metastasis by Apolipoprotein(a) Kringle V in a Nude Mouse Model through the Induction of Apoptosis in Tumor-Associated Endothelial Cells

The formation of liver metastases in colorectal cancer patients is the primary cause of patient death. Current therapies directed at liver metastasis from colorectal cancer have had minimal impact on patient outcomes. Therefore, the development of alternative treatment strategies for liver metastasis is needed. In the present study, we demonstrated that recombinant human apolipoprotein(a) kringle V, also known as rhLK8, induced the apoptotic turnover of endothelial cells in vitro through the mitochondrial apoptosis pathway. The interaction of rhLK8 with glucose-regulated protein 78 (GRP78) may be involved in the induction of apoptosis because the inhibition of GRP78 by GRP78-specific antibodies or siRNA knockdown inhibited the rhLK8-mediated apoptosis of human umbilical vein endothelial cells in vitro. Next, to evaluate the effects of rhLK8 on angiogenesis and metastasis, an experimental model of liver metastasis was established by injecting a human colorectal cancer cell line, LS174T, into the spleens of BALB/c nude mice. The systemic administration of rhLK8 significantly suppressed liver metastasis from human colorectal cancer cells and improved host survival compared with controls. The combination of rhLK8 and 5-fluorouracil substantially increased these survival benefits compared with either therapy alone. Histological observation showed significant induction of apoptosis among tumor-associated endothelial cells in liver metastases from rhLK8-treated mice compared with control mice. Collectively, these results suggest that the combination of rhLK8 with conventional chemotherapy may be a promising approach for the treatment of patients with life-threatening colorectal cancer liver metastases.

The Ubiquitin Ligase ASB4 Promotes Trophoblast Differentiation through the Degradation of ID2

Vascularization of the placenta is a critical developmental process that ensures fetal viability. Although the vascular health of the placenta affects both maternal and fetal well being, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4) promotes embryonic stem cell differentiation to vascular lineages and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2) negatively regulates vascular differentiation during development and is a target of many ubiquitin ligases. Due to their overlapping spatiotemporal expression pattern in the placenta and contrasting effects on vascular differentiation, we investigated whether ASB4 regulates ID2 through its ligase activity in the placenta and whether this activity mediates vascular differentiation. In mouse placentas, ASB4 expression is restricted to a subset of cells that express both stem cell and endothelial markers. Placentas that lack Asb4 display immature vascular patterning and retain expression of placental progenitor markers, including ID2 expression. Using JAR placental cells, we determined that ASB4 ubiquitinates and represses ID2 expression in a proteasome-dependent fashion. Expression of ASB4 in JAR cells and primary isolated trophoblast stem cells promotes the expression of differentiation markers. In functional endothelial co-culture assays, JAR cells ectopically expressing ASB4 increased endothelial cell turnover and stabilized endothelial tube formation, both of which are hallmarks of vascular differentiation within the placenta. Co-transfection of a degradation-resistant Id2 mutant with Asb4 inhibits both differentiation and functional responses. Lastly, deletion of Asb4 in mice induces a pathology that phenocopies human pre-eclampsia, including hypertension and proteinuria in late-stage pregnant females. These results indicate that ASB4 mediates vascular differentiation in the placenta via its degradation of ID2.

Cavernous Hemangioma With Large Phlebolith of the Parotid Gland

Hemangiomas are vascular anomalies characterized by increased proliferation and turnover of endothelial cells. Hemangiomas of the parotid region are relatively uncommon in adult population, and there are a few reports of hemangioma with large phlebolith within the parotid gland. We herein report a case of it. Sialography may be a useful investigation method in the evaluation of radiopaque lesions localized intraglandularly in the parotid area to rule out the sialolith. Cavernous hemangioma with phleboliths should be included in the differential diagnosis of a swelling in the mandibular area.

Adult responses to an ischemic stroke in a rat model of neonatal stress and morphine treatment

Critically ill newborn infants experience stressors that may alter brain development. Using a rodent model, we previously showed that neonatal stress, morphine, and stress plus morphine treatments each influence early gene expression and may impair neurodevelopment and learning behavior. We hypothesized that the combination of neonatal stress with morphine may alter neonatal angiogenesis and/or adult cerebral blood vessel density and thus increase injury after cerebral ischemia in adulthood. To test this, neonatal Lewis rats underwent 8h/d maternal separation, plus morning/afternoon hypoxia exposure and either saline or morphine treatment (2mg/kg s.c.) from postnatal day 3–7. A subset received bromodeoxyuridine to track angiogenesis. Adult brains were stained with collagen IV to quantify cerebral blood vessel density. To examine vulnerability to brain injury, postnatal day 80 adult rats underwent right middle cerebral artery occlusion (MCAO) to produce unilateral ischemic lesions. Brains were removed and processed for histology 48h after injury. Brain injury was assessed by histological evaluation of hematoxylin and eosin, and silver staining. In contrast to our hypothesis, neither neonatal morphine, stress, nor the combination affected cerebral vessel density or MCAO-induced brain injury. Neonatal angiogenesis was not detected in adult rats possibly due to turnover of endothelial cells. Although unrelated to angiogenesis, hippocampal granule cell neurogenesis was detected and there was a trend (P=0.073) toward increased bromodeoxyuridine incorporation in rats that underwent neonatal stress. These findings are discussed in contrast to other data concerning the effects of morphine on cerebrovascular function, and acute effects of morphine on hippocampal neurogenesis.

On the Fabric of the Human Body

With the publication of De Humani Corporis Fabrica in 1543, Andreas Vesalius bestowed some of the greatest advancements of anatomic understanding since the time of Galen, correcting major misconceptions, eg, the notion that the great vessels originated in the liver. It took nearly 1500 years for this evolution in anatomic thinking to transpire. It is simultaneously humbling and invigorating to note that nearly 500 years later the study of anatomy is just as lively and full of evolutionary change. In this issue of Circulation , Kajstura and colleagues1 definitively dispatch the dogma of the heart as a terminally differentiated organ, a concept formulated a mere 40 years earlier, and in so doing completely recalibrate our understanding of cellular homeostasis in the healthy and diseased heart. Article see p 1869 In 1973, Radovan Zak2 of the University of Chicago stated unequivocally, “The adult heart enlarges only by enlargement of its component muscle cells. Division of ventricular muscle cells in mammals is not activated after cardiac injury.” This conclusion was largely consistent with work by Linzbach3 in 1960; however, in this earlier comprehensive histological analysis, there was a suggestion that under certain circumstances the number of cardiac muscle fibers might increase. The Anversa laboratory began suggesting the possibility of cardiomyocyte proliferation in 1980,4 and by 1992, the group began to document clear evidence for replication of cardiac muscle cells in response to physiological stress and myocardial injury in both experimental models5–8 and humans.9,10 These early works included such bold statements as “the recognition of factors controlling myocyte proliferation may provide a new approach for regeneration of damaged and lost myocardium, a phenomenon considered impossible for several decades.” Although some recognized the power and implications of these observations, the Pfeffers' call for open-mindedness …