Tyson N. Kim

Arterial-Venous Segregation by Selective Cell Sprouting: An Alternative Mode of Blood Vessel Formation

Making Split Decisions Development of the vertebrate vasculature has been thought to involve just two mechanisms of blood vessel formation. Herbert et al. (p. 294; see the Perspective by Benedito and Adams) identified a third mechanism in zebrafish in which two distinct, unconnected vessels can be derived from a single precursor vessel. Several vascular endothelial growth factors and signaling pathways, including ephrin and notch signaling, coordinated the sorting and segregation of a mixture of arterial and venous-fated precursor cells into distinct arterial and venous vessels. These findings provide a mechanistic framework for how mixed populations of cells can coordinate their behavior to segregate and form distinct blood vessels. An alternative developmental pathway for vertebrate vasculature segregates a precursor vessel into two separate vessels. Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

Femtosecond laser-drilled capillary integrated into a microfluidic device

Recent growth in microfluidic technology is, to a large extent, driven by soft lithography, a high-throughput fabrication technique where polymer materials, such as poly(dimethyl) siloxane (PDMS), are molded to form microscopic channel networks. Nevertheless, the channel architectures that can be obtained by molding are limited. We address this limitation by using femtosecond laser micromachining to add unmoldable features to the microfluidic devices. We apply laser ablation to drill microcapillaries, with diameters as small as 0.5μm and aspect ratios as high as 800:1, in the walls of molded PDMS channels. Finally, we use a laser-drilled microcapillary to trap a polystyrene bead by suction and hold it against a shear flow.

Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice

Brain arteriovenous malformations (BAVMs) can cause devastating stroke in young people and contribute to half of all hemorrhagic stroke in children. Unfortunately, the pathogenesis of BAVMs is unknown. In this article we show that activation of Notch signaling in the endothelium during brain development causes BAVM in mice. We turned on constitutively active Notch4 (int3) expression in endothelial cells from birth by using the tetracycline-regulatable system. All mutants developed hallmarks of BAVMs, including cerebral arteriovenous shunting and vessel enlargement, by 3 weeks of age and died by 5 weeks of age. Twenty-five percent of the mutants showed signs of neurological dysfunction, including ataxia and seizure. Affected mice exhibited hemorrhage and neuronal cell death within the cerebral cortex and cerebellum. Strikingly, int3 repression resolved ataxia and reversed the disease progression, demonstrating that int3 is not only sufficient to induce, but also required to sustain the disease. We show that int3 expression results in widespread enlargement of the microvasculature, which coincided with a reduction in capillary density, linking vessel enlargement to Notchs known function of inhibiting vessel sprouting. Our data suggest that the Notch pathway is a molecular regulator of BAVM pathogenesis in mice, and offer hope that their regression might be possible by targeting the causal molecular lesion.

Notch4 normalization reduces blood vessel size in arteriovenous malformations.

Normalization of Notch expression restores enlarged blood vessels to microvessels through EphB4-mediated reprogramming of arterial endothelial cells. Reducing Inflation Arteriovenous malformations (AVMs) are a class of vascular abnormalities in which arteries connect directly with veins, thus bypassing the capillary beds and diverting blood flow away from tissues. In these vascular diseases, blood vessels, particularly the veins, become inflated in size and eventually rupture, resulting in hemorrhage and ischemia. AVMs, which can be found in any tissue, are particularly problematic in the brain, where surgical options are limited, and they often result in stroke or death. In a tour-de-force study, Murphy et al. now show that dialing down Notch4 receptor signaling in established AVMs in mouse brain reduces the size of enlarged blood vessels, resulting in restoration of blood flow to capillary beds and the reversal of hypoxia in mouse brain tissue. The Notch receptor is a master regulator of arteriovenous development and is up-regulated in AVMs in human brain. Overexpression of a constitutively active form of Notch4 in endothelial cells lining blood vessel walls is sufficient to induce AVMs in mice. In their new work, Murphy et al. first wanted to establish whether correction of Notch4 signaling could induce the regression of AVMs. Using their mouse brain AVM model, they obtained four-dimensional imaging data of the mouse brain vasculature viewed through a window cut into the cranium with two-photon fluorescence microscopy. When Notch4 signaling was normalized, they found regression of enlarged AVMs, which became similar in size to capillaries. This shrinkage in size enabled blood flow to return to oxygen-deprived tissues in the mouse brain. Surprisingly, the authors discovered that AVM regression was not induced by loss of endothelial cells, thrombotic occlusion, or vessel rupture. Rather, it required reprogramming of arterial endothelial cells in the enlarged AVM vessels to a venous endothelial cell specification. This reprogramming was activated by a decrease in Notch4 receptor signaling, which prompted arterial endothelial cells to start expressing the venous marker EphB4. These findings suggest that strategies to manipulate Notch receptor signaling in blood vessel endothelial cells may help to shrink AVMs and may be a new approach to treating AVMs and other vascular diseases. Abnormally enlarged blood vessels underlie many life-threatening disorders including arteriovenous (AV) malformations (AVMs). The core defect in AVMs is high-flow AV shunts, which connect arteries directly to veins, “stealing” blood from capillaries. Here, we studied mouse brain AV shunts caused by up-regulation of Notch signaling in endothelial cells (ECs) through transgenic expression of constitutively active Notch4 (Notch4*). Using four-dimensional two-photon imaging through a cranial window, we found that normalizing Notch signaling by repressing Notch4* expression converted large-caliber, high-flow AV shunts to capillary-like vessels. The structural regression of the high-flow AV shunts returned blood to capillaries, thus reversing tissue hypoxia. This regression was initiated by vessel narrowing without the loss of ECs and required restoration of EphB4 receptor expression by venous ECs. Normalization of Notch signaling resulting in regression of high-flow AV shunts, and a return to normal blood flow suggests that targeting the Notch pathway may be useful therapeutically for treating diseases such as AVMs.

Molecular identification of venous progenitors in the dorsal aorta reveals an aortic origin for the cardinal vein in mammals

Coordinated arterial-venous differentiation is crucial for vascular development and function. The origin of the cardinal vein (CV) in mammals is unknown, while conflicting theories have been reported in chick and zebrafish. Here, we provide the first molecular characterization of endothelial cells (ECs) expressing venous molecular markers, or venous-fated ECs, within the emergent dorsal aorta (DA). These ECs, expressing the venous molecular markers Coup-TFII and EphB4, cohabited the early DA with ECs expressing the arterial molecular markers ephrin B2, Notch and connexin 40. These mixed ECs in the early DA expressed either the arterial or venous molecular marker, but rarely both. Subsequently, the DA exhibited uniform arterial markers. Real-time imaging of mouse embryos revealed EC movement from the DA to the CV during the stage when venous-fated ECs occupied the DA. We analyzed mutants for EphB4, which encodes a receptor tyrosine kinase for the ephrin B2 ligand, as we hypothesized that ephrin B2/EphB4 signaling may mediate the repulsion of venous-fated ECs from the DA to the CV. Using an EC quantification approach, we discovered that venous-fated ECs increased in the DA and decreased in the CV in the mutants, whereas the rest of the ECs in each vessel were unaffected. This result suggests that the venous-fated ECs were retained in the DA and missing in the CV in the EphB4 mutant, and thus that ephrin B2/EphB4 signaling normally functions to clear venous-fated ECs from the DA to the CV by cell repulsion. Therefore, our cellular and molecular evidence suggests that the DA harbors venous progenitors that move to participate in CV formation, and that ephrin B2/EphB4 signaling regulates this aortic contribution to the mammalian CV.

Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals.

Background The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions. Methodology/Principal Findings We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 µm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters. Conclusions/Significance To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies.

Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels

Significance Brain arteriovenous malformations are focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. They can cause ischemia, hemorrhage, disability, and death, particularly in young people, accounting for 50% of childhood stroke. The molecular etiology of the disease remains poorly understood, hindering the development of therapeutic treatments. Here, we report that, in an animal model, the lesion arises from the enlargement of capillary-like vessels. Notch signaling in the endothelium of microvasculature and veins is critical for the disease initiation by increasing cell areas but not proliferation. Blood flow mediates disease progression by a positive feedback of increasing flow and vessel diameter. Our data shed light on the mechanism underlying the pathogenesis of this devastating disease. Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.

Spherical aberration correction in nonlinear microscopy and optical ablation using a transparent deformable membrane

We describe the design and utilization of a deformable membrane to minimize the negative spherical aberration that occurs when a standard water-dipping objective is used to focus within a higher-index sample. In connection with two-photon laser scanning microscopy, we demonstrate twofold improved axial resolution of structures as deep as 1mm in gels and brain tissue. In conjunction with plasma-mediated ablation, we demonstrate enhanced production of optical damage deep within a glass substrate. The present method provides a simple and inexpensive correction for a limited yet important class of optical aberrations.

Active cathepsins B, L, and S in murine and human pancreatitis.

Cathepsins regulate premature trypsinogen activation within acinar cells, a key initial step in pancreatitis. The identity, origin, and causative roles of activated cathepsins in pancreatic inflammation and pain are not defined. By using a near infrared-labeled activity-based probe (GB123) that covalently modifies active cathepsins, we localized and identified activated cathepsins in mice with cerulein-induced pancreatitis and in pancreatic juice from patients with chronic pancreatitis. We used inhibitors of activated cathepsins to define their causative role in pancreatic inflammation and pain. After GB123 administration to mice with pancreatitis, reflectance and confocal imaging showed significant accumulation of the probe in inflamed pancreas compared with controls, particularly in acinar cells and macrophages, and in spinal cord microglia and neurons. Biochemical analysis of pancreatic extracts identified them as cathepsins B, L, and S (Cat-B, Cat-L, and Cat-S, respectively). These active cathepsins were also identified in pancreatic juice from patients with chronic pancreatitis undergoing an endoscopic procedure for the treatment of pain, indicating cathepsin secretion. The cathepsin inhibitor K11777 suppressed cerulein-induced activation of Cat-B, Cat-L, and Cat-S in the pancreas and ameliorated pancreatic inflammation, nocifensive behavior, and activation of spinal nociceptive neurons. Thus pancreatitis is associated with an increase in the active forms of the proteases Cat-B, Cat-L, and Cat-S in pancreatic acinar cells and macrophages, and in spinal neurons and microglial cells. Inhibition of cathepsin activation ameliorated pancreatic inflammation and pain. Activity-based probes permit identification of proteases that are predictive biomarkers of disease progression and response to therapy and may be useful noninvasive tools for the detection of pancreatic inflammation.

Synchronous ipsilateral Bochdalek and Morgagni diaphragmatic hernias: a case report

The etiology of congenital diaphragmatic hernia (CDH) is unknown. Phenotypic patterns of CDH defects provide clues about normal diaphragm development and the pathophysiology of CDH. We report a case of a patient who was diagnosed with CDH postnatally and was found on imaging to have simultaneous Bochdalek and Morgagni hernias on the right side. During the operative repair of these defects, an additional left-sided Morgagni-type defect was also found. To the best of our knowledge, this form of CDH has not been previously reported.