Search

The number of coronavirus disease 2019 (COVID‐19) cases has been increasing significantly, and the disease has evolved into a global pandemic, posing an unprecedented challenge to the healthcare community. Angiotensin‐converting enzyme 2, the binding and entry receptor of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in hosts, is also expressed on pulmonary vascular endothelium; thus, pulmonary vasculature is a potential target in COVID‐19. Indeed, pulmonary vascular thickening is observed by early clinical imaging, implying a tropism of SARS‐CoV‐2 for pulmonary vasculature. Recent studies reported that COVID‐19 is associated with vascular endothelial damage and dysfunction along with inflammation, coagulopathy, and microthrombosis; all of these pathologic changes are the hallmarks of pulmonary vascular diseases. Notwithstanding the not fully elucidated effects of COVID‐19 on pulmonary vasculature, the vascular endotheliopathy that occurs after infection is attributed to direct infection and indirect damage mainly caused by renin‐angiotensin‐aldosterone system imbalance, coagulation cascade, oxidative stress, immune dysregulation, and intussusceptive angiogenesis. Degradation of endothelial glycocalyx exposes endothelial cell (EC) surface receptors to the vascular lumen, which renders pulmonary ECs more susceptible to SARS‐CoV‐2 infection. The present article reviews the potential pulmonary vascular pathophysiology and clinical presentations in COVID‐19 to provide a basis for clinicians and scientists, providing insights into the development of therapeutic strategies targeting pulmonary vasculature., SARS‐CoV‐2 infection‐induced pulmonary vascular injury and potential treatments target the pulmonary vascular system.

Key points Chronic fetal hypoxia is one of the most common complications of pregnancy and is known to cause fetal growth restriction. The structural adaptations of the placental vasculature responsible for growth restriction with chronic hypoxia are not well elucidated. Using a mouse model of chronic maternal hypoxia in combination with micro-computed tomography and scanning electron microscopy, we found several placental adaptations that were beneficial to fetal growth including capillary expansion, thinning of the interhaemal membrane and increased radial artery diameters, resulting in a large drop in total utero-placental vascular resistance. One of the mechanisms used to achieve the rapid increase in capillaries was intussusceptive angiogenesis, a strategy used in human placental development to form terminal gas-exchanging villi. These results contribute to our understanding of the structural mechanisms of the placental vasculature responsible for fetal growth restriction and provide a baseline for understanding adaptive physiological responses of the placenta to chronic hypoxia. Abstract The fetus and the placenta in eutherian mammals have a unique set of compensatory mechanisms to respond to several pregnancy complications including chronic maternal hypoxia. This study examined the structural adaptations of the feto- and utero-placental vasculature in an experimental mouse model of chronic maternal hypoxia (11% O2 from embryonic day (E) 14.5-E17.5). While placental weights were unaffected by exposure to chronic hypoxia, using micro-computed tomography, we found a 44% decrease in the absolute feto-placental arterial vascular volume and a 30% decrease in total vessel segments in the chronic hypoxia group compared to control group. Scanning electron microscopy imaging showed significant expansion of the capillary network; consequently, the interhaemal membrane was 11% thinner to facilitate maternal-fetal exchange in the chronic hypoxia placentas. One of the mechanisms for the rapid capillary expansion was intussusceptive angiogenesis. Analysis of the utero-placental arterial tree showed significant increases (24%) in the diameter of the radial arteries, resulting in a decrease in the total utero-placental resistance by 2.6-fold in the mice exposed to chronic maternal hypoxia. Together these adaptations acted to preserve placental weight whereas fetal weight was decreased.

Recently, we demonstrated that in human intravascular papillary endothelial hyperplasia (IPEH), vein wall vascularization occurs in association with myriad papillae, a large part of which formed in the vascularized vein wall. Previously, using an animal model, we observed that PGE2 and glycerol administration around the femoral vein originates intense vascularization of the vein wall from its intimal endothelial cells (ECs). This vascularization is similar to that in IPEH. The aim of this study is to assess the mechanism of papillary formation, using this model after demonstrating papillary development in neo-vascularized femoral vein walls. In semithin and ultrathin sections, the sequential vascular and papillary development was as follows: (a) activation of vein intimal ECs, (b) sprouting of intimal ECs towards the vein media layer and microvessel development, (c) interconnection between neighboring microvessels originated elementary loops, which encircled vein wall components and formed papillae. The encircling ECs formed the papillary cover, and the encircled component formed the core. The papillae showed a similar structure to that of folds and pillars in intussusceptive angiogenesis, and (d) origin of secondary and complex loop systems by interconnection of neighboring elementary loops and by splitting of papillae by new loops, with abundant papillary development. In conclusion, the results support a piecemeal angiogenic mechanism in papillary formation, with association of sprouting and intussusceptive types of angiogenesis. Further studies are needed to assess whether the intravascular papillae described in several pathologic processes, including vessel tumors, such as Dabska's tumor, retiform hemangioendothelioma, and angiosarcoma, follow a similar mechanism. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1781-1792, 2017. © 2017 Wiley Periodicals, Inc.

The conventional view of tumour vascularization is that tumours acquire their blood supply from neighbouring normal stroma. Additional methods of tumour vascularization such as intussusceptive angiogenesis, vasculogenic mimicry, vessel co-option and vasculogenesis have been demonstrated to occur. However, the origin of the endothelial cells and pericytes in the tumour vasculature is not fully understood. Their origin from malignant cells has been shown indirectly in lymphoma and neuroblastoma by immuno-FISH experiments. It is now evident that tumours arise from a small population of cells called cancer stem cells (CSCs) or tumour initiating cells. Recent data suggest that a proportion of tumour endothelial cells arise from cancer stem cells in glioblastoma. This was demonstrated both in vitro and in vivo. The analysis of chromosomal abnormalities in endothelial cells showed identical genetic changes to those identified in tumour cells. However, another report contradicted these results from the earlier studies in glioblastoma and had shown that CSCs give rise to pericytes and not endothelial cells. The main thrust of this review is the critical analysis of the conflicting data from different studies and the remaining questions in this field of research. The mechanism by which this phenomenon occurs is also discussed in detail. The transdifferentiation of CSCs to endothelial cells/pericytes has many implications in the progression and metastasis of the tumours and hence it would be a novel target for antiangiogenic therapy.

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best‐understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine‐tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF‐R2 activation by VEGF nor its internalization, but it modulates VEGF‐R2 downstream signaling through phospho‐ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF‐induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.

Summary The word angiogenesis indicates the formation of new vascular segments from existing vessels such as capillaries and venules. Blood vessel formation in tumors is the result of rapid, disorganized vascular growth through two distinct mechanisms: sprouting and intussusceptive angiogenesis. The objective of this study was to elucidate the morphological aspects of these two vascular growth mechanisms in oral squamous cell carcinoma induced in hamster buccal pouch. Eight Syrian golden hamsters had their right buccal pouch treated with DMBA 0.5% and 10% carbamide peroxide for 90 days in order to produce squamous cell carcinoma in this site. Next, buccal pouches of the animals were submitted to the vascular corrosion technique and then analyzed by scanning electron microscopy. The vascular figures of sprouts were observed in the entire vascular network of the buccal pouches, as opposed to the intussusceptive angiogenesis that was predominantly observed in the sub-epithelial network. It was possible to differentiate the figures of sprouts from artifacts by the analysis of the blind ending of these structures. Intussusceptive angiogenesis was identified by the presence of holes trespassing the lumen of the capillaries. Vascular expansion occurred through intussusceptive angiogenesis in two ways: by the fusion of the pillars to form a new capillary and, by increasing the girth of the pillar to form meshes. The method of corrosion associated with scanning electron microscopy proved to be an excellent tool to study the two types of angiogenesis in oral squamous cell carcinoma in the hamster buccal pouch. SCANNING 36:293–300, 2014. © 2013 Wiley Periodicals, Inc.

Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention.

Angiogenesis is essential for the growth of primary tumors and for their metastasis. This process is induced by factors, such as vascular endothelial growth factors (VEGFs), that bind to transmembrane VEGF receptors (VEGFRs). VEGF-A is the primary factor involved with angiogenesis; it binds to both VEGFR-1 and VEGFR-2. The inhibition of angiogenesis by obstructing VEGF-A signaling has been investigated as a method to treat solid tumors, but the development of resistance to this blockade has complicated treatment. The major mechanisms of this resistance to VEGF-A blockade include signaling by redundant receptors, such as the fibroblast growth factors, angiopoietin-1, ephrins, and other forms of VEGF. Other major mechanisms of resistance are increased metastasis of hypoxia-resistant tumor cells, recruitment of cell types capable of promoting VEGF-independent angiogenesis, and increased circulation of nontumor proangiogenic factors. Additional mechanisms of resistance to VEGF-A blockade include heterogeneity of responsiveness among tumor cells, use of anti-VEGF-A agents at insufficient doses or for insufficient duration, altered sensitivity to anti-VEGF-A agents by mutations in endothelial cells or vascular remodeling, maintenance of vascular sleeves that allow for easy regrowth of tumor vasculature upon discontinuation of therapy, vascular cooption, and intussusceptive angiogenesis. An understanding of these mechanisms may lead to the development of targeted therapies that overcome this resistance. Some of these approaches include the combined inhibition of redundant angiogenic pathways, proper patient selection for various therapies based on gene expression profiles, blockade of cellular migration by inhibition of colony-stimulating factor, or the use of agents to disrupt vascular architecture. Cancer 2012;3455–3467. © 2011 American Cancer Society.

Adaptation of vascular networks to functional demands needs vessel growth, vessel regression and vascular remodelling. Biomechanical forces resulting from blood flow play a key role in these processes. It is well-known that metabolic stimuli, mechanical forces and flow patterns can affect gene expression and remodelling of vascular networks in different ways. For instance, in the sprouting type of angiogenesis related to hypoxia, there is no blood flow in the rising capillary sprout. In contrast, it has been shown that an increase of wall shear stress initiates the splitting type of angiogenesis in skeletal muscle. Otherwise, during development, both sprouting and intussusception act in parallel in building the vascular network, although with differences in spatiotemporal distribution. Thereby, in addition to regulatory molecules, flow dynamics support the patterning and remodelling of the rising vascular tree. Herewith, we present an overview of angiogenic processes with respect to intussusceptive angiogenesis as related to local haemodynamics.

Intussusceptive angiogenesis is a morphogenetic process that forms new blood vessels by the division of a single blood vessel into two lumens. Here, we show that this process of intraluminal division participates in the inflammation-induced neovascularization associated with chemically induced murine colitis. In studies of both acute (4-7 days) and chronic (28-31 days) colitis, intravital microscopy of intravascular tracers demonstrated a twofold reduction in blood flow velocity. In the acute colitis model, the decreased velocity was associated with marked dilatation of the mucosal plexus. In contrast, chronic inflammation was associated with normal caliber vessels and duplication (and triplication) of the quasi-polygonal mucosal plexus. Scanning electron microscopy (SEM) of intravascular corrosion casts suggested that pillar formation and septation, previously linked to the morphogenetic process of intussusceptive angiogenesis, were present within days of the onset of inflammation. Four weeks after the onset of inflammation, SEM of vascular corrosion casts demonstrated replication of the mucosal plexus without significant evidence of sprouting angiogenesis. These data suggest that mucosal capillaries have comparable aggregate cross-sectional area in acute and chronic colitis; however, there is a significant increase in functional capillary density in chronic colitis. We conclude that intussusceptive angiogenesis is a fundamental mechanism of microvascular adaptation to prolonged inflammation.

Embryonic development is associated with extensive vascular growth and remodeling. We used immunohistochemical, light and electron microscopical techniques, as well as vascular casting methods to study the developing chick embryo kidney with special attention to the interplay between sprouting and intussusceptive vascular growth modes. During inauguration at embryonic day 5 (E5), the early mesonephros was characterised by extensive microvascular sprouting. By E7, the vascular growth mode switched to intussusception, which contributed to rapid kidney vasculature growth up to E11, when the first obvious signs of vascular degeneration were evident. The metanephros underwent similar phases of vascular development inaugurating at E8 with numerous capillary sprouts and changing at E13 to intussusceptive growth, which was responsible for vascular amplification and remodeling. A phenomenal finding was that future renal lobules arose as large glomerular tufts, supplied by large vessels, which were split into smaller intralobular feeding and draining vessels with subsequent formation of solitary glomeruli. This glomerular duplication was achieved by intussusception, i.e., by formation of pillars in rows and their successive merging to delineate the vascular entities. Ultimately, the maturation of the vasculature was achieved by intussusceptive pruning and branching remodeling. An interesting finding was that strong VEGF expression was associated with the sprouting phase of angiogenesis while bFGF was upregulated during the phase of intussusceptive microvascular growth. We conclude that microvascular growth and remodeling in avian kidney follows an adroitly crafted pattern, which entails a precise spaciotemporal interplay between sprouting and intussusceptive angiogenic growth modes supported partly by VEGF and bFGF.

The theory of bifurcating vascular systems predicts vessel diameters that are related to optimality criteria like minimization of pumping energy or of building material. However, mechanisms for producing the postulated optimality have not been described so far, and quantitative data on bifurcation diameters during development are scarce. We used an embryonic vascular bed that rapidly grows and adapts to changing hemodynamic conditions, the chicken chorioallantoic membrane (CAM), and correlated vascular cast and tissue section morphology with in vivo time-lapse video monitoring. The bifurcation exponent Δ and associated parameters were quantitatively assessed in arterial and venous microvessels ranging in diameter from 30 to 100 μm. We observed emergence of optimality by means of intussusception, i.e., formation of transvascular tissue pillars. In addition to intussusceptive microvascular growth (IMG = expansion of capillary networks) and intussusceptive arborization (IAR = formation of feeding vessels from capillaries) the observed intussusception at bifurcations represents a third variant of nonsprouting angiogenesis. We call it intussusceptive branching remodeling (IBR). IBR occurred in vessels of considerable diameter by means of two alternative mechanisms: either through pillars arising close to a bifurcation, which increased in girth until they merged with the connective tissue in the bifurcation angle; or through pillars arising at some distance from the bifurcation point, which then expanded by formation of ingrowing tissue folds until they became connected to the tissue of the bifurcation angle. Morphologic evidence suggests that IBR is a wide-spread phenomenon, taking place also in lung, intestinal, kidney, eye, etc., vasculature. Irrespective of the mode followed, IBR led to a branching pattern close to the predicted optimum, Δ = 3.0. Significant differences were observed between Δ at arterial bifurcations (2.70 to 2.90) and Δ at venous bifurcations (2.93 to 3.75). IBR, by means of eccentric pillar formation and fusion, was also involved in vascular pruning. Experimental changes in CAM hemodynamics (by locally increasing blood flow) induced onset of IBR within less than 1 hr. Our study provides morphologic and quantitative evidence that a similar cellular machinery is used for all three variants of vascular intussusception, IMG, IAR, and IBR. It thus provides a mechanism of efficiently generating complex blood transport systems from limited genetic information. Differential quantitative outcome of IBR in arteries and veins, and the experimental induction of IBR strongly suggest that hemodynamic factors can instruct embryonic vascular remodeling toward optimality. © 2002 Wiley-Liss, Inc.

Angiogenesis is the emergence of new blood and lymph vessels from existing ones. In the pathologic form it contributes to the onset and progression of numerous different human disorders such as cancer, inflammation, atherosclerosis and blinding eye diseases. There exist a number of models to study angiogenesis, both in vitro and in vivo, but there is no single perfect model so far. Consequently there is a need to develop new angiogenesis assays for evaluating blood and lymph vessel behaviour in different physiologic settings.The aim of this thesis was to gain insight into in vivo angiogenesis introducing a new technique in an inflammatory corneal model. The method involved in vivo examination of the cornea and subsequent comparison of in vivo findings with ex vivo immunohistochemical analysis of the same tissue samples. An existing suture model for inflammatory angiogenesis in the cornea was modified for in vivo observations with a clinically-approved corneal confocal microscope.In this thesis, corneal lymph vessels were characterized for the first time in vivo and findings from the experimental bench could be applied in a clinical setting, where presumed lymphatics were observed in a corneal transplant patient with rejection. Furthermore, the technique was extended to investigate time-lapse processes in sprouting and regressing capillaries, and led to a number of new observations. CD11b+ myeloid cells constitute the first bulk of infiltrating inflammatory cells and contribute to inflammatory sprouting and regression in numerous ways including pre-patterning of the corneal stroma and guiding of capillary sprouts. Newly formed hemangiogenic sprouts are perfused with a slow-moving fluid and have a lumen. In blood vessel regression, capillary remodeling occurred by abandonment of sprout tips in close association with macrophages and vascular loops formed by presumed intussusceptive angiogenesis. In addition, a network of pericyte- and endothelium-free basement membrane tubes was formed after desertion or degradation of vascular endothelium in former corneal capillaries.In conclusion, we introduce a new in vivo technique for investigating angiogenesis in a corneal model were in vivo findings can be interpreted with ex vivo definitions of specific cell types by immunohistochemistry. Findings from pre-clinical experiments have been possible to apply in a clinical setting when examining patients with corneal pathology.

The mammary gland life cycle is exemplified by massive, physiologically dictated changes in cell number and composition, architecture, and functionality. These drastic upheavals, by necessity, also involve the mammary endothelium, which undergoes angiogenic expansion during pregnancy and lactation followed by ordered regression during involution. In this review, we summarise data obtained using the Mercox methyl methacrylate corrosion cast technique to analyse the mammary gland vasculature during normal development and carcinogenesis. Concomitant with epithelial cell expansion, the mammary vasculature grows during the first half of pregnancy by sprouting angiogenesis whereas the last half of pregnancy and lactation are characterised by the non-proliferative intussusceptive angiogenesis. The vasculature of the lactating gland is composed of a well-developed capillary meshwork enveloping the secretory alveoli with basket-like honeycomb structures. During involution, regression of the vasculature is achieved by regional collapse of the honeycomb structures, capillary retraction, and endothelial attenuation. This process appears partly to involve apoptosis. However, an additional mechanism involving remodelling without cell death, which we have termed angiomeiosis, must exist to explain the morphological observations. Interestingly, in mammary tumours of neuT transgenic mice, both sprouting and intussusceptive angiogenesis was observed simultaneously in the same nodules, a finding with potential implications for cancer therapy. The underlying molecular mechanisms controlling angiogenic modulation in the mammary gland, particularly angiogenic regression and the endothelial:parenchymal interplay, are poorly understood. However, the data summarised in this review indicate that precisely these molecular mechanisms offer novel alternatives for specific and effective treatment of breast cancer. Microsc. Res. Tech. 52:182–189, 2001. © 2001 Wiley-Liss, Inc.

To assess the impact of vascular endothelial growth factor (VEGF) on intussusceptive angiogenesis.Polyurethane casts of the microvasculature of chicken chorioallantoic membrane (CAM) were prepared on embryonic days (E) 8, 10, 12, and 14. At light microscopy level, minute holes (2 microm in diameter) and hollows (2 microm) were observed in the casts. Transmission electron microscopy disclosed the minute holes to mainly represent transluminal pillars characteristic for intussusceptive angiogenesis. The numerical density of the holes/pillars was highest at an early (E8) and a late (E12-E14) stage. Only mRNA of VEGF-A-122 and VEGF-A-166 isoforms was detected in the CAM. The transcription rate of VEGF-A mRNA peaked on E8/9 and E12, while VEGF-A protein expression increased on E8/9 and E11/12 to rapidly decrease thereafter as determined by immunoblotting. At all time points investigated, VEGF-A immunohistochemical reactivity was restricted to cells of the chorionic epithelium in direct contact to the capillary plexus. When the VEGF-R-inhibitor PTK787/ZK222584 (0.1 mg/mL) was applied on E9 CAM, the microvasculature topology on E12 was similar to that on E10.The temporal course of intussusception corresponded to the expression of VEGF-A in CAM microvasculature. Inhibition of VEGF-signaling retarded intussusceptive-dependent capillary maturation. These data suggest that VEGF promotes intussusception.

The aim of the present study was to verify the occurrence of intussusceptive angiogenesis in blood vessels from submandibular lymph nodes responsible for lymphatic drainage of the tongue. A surgical wound inflicted on the ventral tongue of male Wistar rats and submandibular regional lymph nodes were evaluated at different postoperative periods. Scanning electron microscopy (SEM) was used to observe 123 lymph nodes at times 2, 3, 7, 10, 14, and 21 postoperative days. During the analysis of the vascular models with SEM, intussusceptive angiogenesis was observed in all groups evaluated. This was more extensive on the second and third postoperative days (83.33% and 80%, respectively), representing in these groups the expansion of the vascular chain of lymph nodes. At 21 postoperative days, intussusceptive angiogenesis (42.85%) was suggestive of vascular remodeling. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.

During postnatal growth the parenchymal septa of rat lung undergo an impressive restructuring. While immature septa are thick and contain two capillary layers, mature septa are slender and contain a single microvascular network. Using the Mercox casting technique and scanning electron microscopy, we investigated the mode and the timing of the transformation of the pulmonary capillary bed. During the third postnatal week the parenchymal septa rapidly mature to match adult morphology. Even in adult lungs, however, remnants of the immature status are present: A capillary bilayer is regularly found at the base and the tip of the septa. Our observations support the concept that reduction of intervening tissue, partial fusion of the two capillary networks, and preferential growth lead to the mature vascular arrangement. The fact that true mature interalveolar septa show a denser capillary network than alveolar walls abutting onto pleura, bronchi, or larger vessels is consonant with the fusion theory. Towards the nonparenchyma, the capillary network surrounding every airspace had no counterpart to fuse with. From quantitative data it can be calculated that owing to lung growth, mesh size should increase more than four times between birth and adult age. The adult lung network, however, is denser than the one in young animals. This means that new meshes must be added during growth. We propose that small holes observed in sheet-like regions of the microvasculature enlarge to form new capillary meshes. With this mechanism of in-itself or intussusceptional growth, sprouting of individual capillary segments to increase network size is no longer needed.