Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice.

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

Nuclear Mechanosensation and Mechanotransduction in Vascular Cells.

Vascular cells are constantly subjected to physical forces associated with the rhythmic activities of the heart, which combined with the individual geometry of vessels further imposes oscillatory, turbulent, or laminar shear stresses on vascular cells. These hemodynamic forces play an important role in regulating the transcriptional program and phenotype of endothelial and smooth muscle cells in different regions of the vascular tree. Within the aorta, the lesser curvature of the arch is characterized by disturbed, oscillatory flow. There, endothelial cells become activated, adopting pro-inflammatory and athero-prone phenotypes. This contrasts the descending aorta where flow is laminar and endothelial cells maintain a quiescent and atheroprotective phenotype. While still unclear, the specific mechanisms involved in mechanosensing flow patterns and their molecular mechanotransduction directly impact the nucleus with consequences to transcriptional and epigenetic states. The linker of nucleoskeleton and cytoskeleton (LINC) protein complex transmits both internal and external forces, including shear stress, through the cytoskeleton to the nucleus. These forces can ultimately lead to changes in nuclear integrity, chromatin organization, and gene expression that significantly impact emergence of pathology such as the high incidence of atherosclerosis in progeria. Therefore, there is strong motivation to understand how endothelial nuclei can sense and respond to physical signals and how abnormal responses to mechanical cues can lead to disease. Here, we review the evidence for a critical role of the nucleus as a mechanosensor and the importance of maintaining nuclear integrity in response to continuous biophysical forces, specifically shear stress, for proper vascular function and stability.

Aortic intimal resident macrophages are essential for maintenance of the non-thrombogenic intravascular state.

Leukocytes and endothelial cells frequently cooperate to resolve inflammatory events. In most cases, these interactions are transient in nature and triggered by immunological insults. Here, we report that in areas of disturbed blood flow, aortic endothelial cells permanently and intimately associate with a population of specialized macrophages that are recruited at birth from the closing ductus arteriosus and share the luminal surface with the endothelium becoming interwoven in the tunica intima. Anatomical changes that affect hemodynamics, like in patent ductus arteriosus, alter macrophage seeding to coincide with regions of disturbed flow. Aortic resident macrophages expand in situ via direct cell renewal. Induced-depletion of intimal macrophages led to thrombin-mediated endothelial cell contraction, progressive fibrin accumulation and formation of microthrombi that, once dislodged, caused blockade of vessels in several organs. Together the findings revealed that intravascular resident macrophages are essential to regulate thrombin activity and clear fibrin deposits in regions of disturbed blood flow.

Transcriptional Evaluation of the Ductus Arteriosus at the Single-Cell Level Uncovers a Requirement for Vim (Vimentin) for Complete Closure.

OBJECTIVE: Failure to close the ductus arteriosus, patent ductus arteriosus, accounts for 10% of all congenital heart defects. Despite significant advances in patent ductus arteriosus management, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required. METHODS: We performed single-cell RNA-sequencing of the ductus arteriosus in mouse embryos at E18.5 (embryonic day 18.5), and P0.5 (postnatal day 0.5), and P5 to identify transcriptional alterations that might be associated with remodeling. We further confirmed our findings using transgenic mouse models coupled with immunohistochemistry analysis. RESULTS: The intermediate filament vimentin emerged as a candidate that might contribute to closure of the ductus arteriosus. Indeed, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus. To seek mechanisms, we turned to the RNA-sequencing data that indicated changes in Jagged1 with similar profile to vimentin and pointed to potential links with Notch. In fact, Notch3 signaling was impaired in vimentin null mice and vimentin null mice phenocopies patent ductus arteriosus in Jagged1 endothelial and smooth muscle deleted mice. CONCLUSIONS: Through single-cell RNA-sequencing and by tracking closure of the ductus arteriosus in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus through a mechanism that includes deregulation of the Notch signaling pathway.

Selective and Marked Blockade of Endothelial Sprouting Behavior Using Paclitaxel and Related Pharmacologic Agents.

Whether alterations in the microtubule cytoskeleton affect the ability of endothelial cells (ECs) to sprout and form branching networks of tubes was investigated in this study. Bioassays of human EC tubulogenesis, where both sprouting behavior and lumen formation can be rigorously evaluated, were used to demonstrate that addition of the microtubule-stabilizing drugs, paclitaxel, docetaxel, ixabepilone, and epothilone B, completely interferes with EC tip cells and sprouting behavior, while allowing for EC lumen formation. In bioassays mimicking vasculogenesis using single or aggregated ECs, these drugs induce ring-like lumens from single cells or cyst-like spherical lumens from multicellular aggregates with no evidence of EC sprouting behavior. Remarkably, treatment of these cultures with a low dose of the microtubule-destabilizing drug, vinblastine, led to an identical result, with complete blockade of EC sprouting, but allowing for EC lumen formation. Administration of paclitaxel in vivo markedly interfered with angiogenic sprouting behavior in developing mouse retina, providing corroboration. These findings reveal novel biological activities for pharmacologic agents that are widely utilized in multidrug chemotherapeutic regimens for the treatment of human malignant cancers. Overall, this work demonstrates that manipulation of microtubule stability selectively interferes with the ability of ECs to sprout, a necessary step to initiate and form branched capillary tube networks.

Electrochemical cytosensors for detection of breast cancer cells.

Breast cancer is one of lethal cancers among women with its metastasis leading to cancer-related morbidity and mortality. Circulating tumor cells (CTCs) derived from a primary tumor can be detected in the venous blood of cancer patients. Monitoring CTCs in blood samples has increased exponentially over the past decades and holds great promise in the diagnosis and treatment of metastatic breast cancer. Electrochemical cytosensors, classified as a class of electrochemical biosensors for sensitive detection and enumeration of targeted cells with minimally invasive methods, have the advantages of electrochemical biosensors, such as simplicity, low cost, and low limit of detection. Here, we review recent progress in the detection of CTCs from breast cancer with a focus on electrochemical cytosensors. This review describes platforms benefiting from these cytosensors to identify cancerous breast cells. Furthermore, strategies for signal amplification and also generation of reusable electrochemical cytosensors are introduced. In addition, breast cancer markers and biorecognition elements for cell capturing are reviewed.

Evaluation and Characterization of Endothelial Cell Invasion and Sprouting Behavior.

Here, we describe highly reproducible methods to investigate human EC invasion and sprouting behavior in 3D collagen matrices. Two assay models are presented whereby ECs are induced to sprout from a monolayer surface or from aggregated ECs suspended within a collagen gel matrix. In each case, the assays are performed using serum-free defined media containing a combination of five growth factors (Factors): FGF-2, SCF, IL-3, SDF-1α, and insulin. In both models, marked EC sprouting occurs with leading EC tip cells over a 12-24 h period. To illustrate their utility, we present data showing the influence of various pharmacologic inhibitors directed to membrane-type matrix metalloproteinases (MT-MMPs), protein kinase C alpha (PKCα), Src family kinases, and Notch-dependent signaling. Marked inhibition of sprouting is observed after blockade of MT-MMPs and PKCα, while strong increases in sprouting and EC tip cell number is observed following blockade of Src kinases, Notch signaling or both. Interestingly, the increased sprouting behavior observed following Src or Notch blockade directly correlates with a loss in the ability of ECs to form lumens. These defined in vitro assay models allow for a genetic and signaling dissection of EC tip cells vs. lumen forming ECs, which are both necessary for the formation of branching networks of tubes during vascular morphogenic events.

Src- and Fyn-dependent apical membrane trafficking events control endothelial lumen formation during vascular tube morphogenesis.

Here we examine the question of how endothelial cells (ECs) develop their apical membrane surface domain during lumen and tube formation. We demonstrate marked apical membrane targeting of activated Src kinases to this apical domain during early and late stages of this process. Immunostaining for phosphotyrosine or phospho-Src reveals apical membrane staining in intracellular vacuoles initially. This is then followed by vacuole to vacuole fusion events to generate an apical luminal membrane, which is similarly decorated with activated phospho-Src kinases. Functional blockade of Src kinases completely blocks EC lumen and tube formation, whether this occurs during vasculogenic tube assembly or angiogenic sprouting events. Multiple Src kinases participate in this apical membrane formation process and siRNA suppression of Src, Fyn and Yes, but not Lyn, blocks EC lumen formation. We also demonstrate strong apical targeting of Src-GFP and Fyn-GFP fusion proteins and increasing their expression enhances lumen formation. Finally, we show that Src- and Fyn-associated vacuoles track and fuse along a subapically polarized microtubule cytoskeleton, which is highly acetylated. These vacuoles generate the apical luminal membrane in a stereotypically polarized, perinuclear position. Overall, our study identifies a critical role for Src kinases in creating and decorating the EC apical membrane surface during early and late stages of lumen and tube formation, a central event in the molecular control of vascular morphogenesis.

Histologic changes associated with talaporfin sodium-mediated photodynamic therapy in rat skin.

BACKGROUND AND OBJECTIVE: Alternative treatments are needed to achieve consistent and more complete port wine stain (PWS) removal, especially in darker skin types; photodynamic therapy (PDT) is a promising alternative treatment. To this end, we previously reported on Talaporfin Sodium (TS)-mediated PDT. It is essential to understand treatment tissue effects to design a protocol that will achieve selective vascular injury without ulceration and scarring. The objective of this work is to assess skin changes associated with TS-mediated PDT with clinically relevant treatment parameters. STUDY DESIGN/MATERIALS AND METHODS: We performed TS (0.75 mg/kg)-mediated PDT (664 nm) on Sprague Dawley rats. Radiant exposures were varied between 15 and 100 J/cm2 . We took skin biopsies from subjects at 9 hours following PDT. We assessed the degree and depth of vascular and surrounding tissue injury using histology and immunohistochemical staining. RESULTS: TS-mediated PDT at 0.75 mg/kg combined with 15 and 25 J/cm2 light doses resulted in vascular injury with minimal epidermal damage. At light dose of 50 J/cm2 , epidermal damage was noted with vascular injury. At light doses >50 J/cm2 , both vascular and surrounding tissue injury were observed in the forms of vasculitis, extravasated red blood cells, and coagulative necrosis. Extensive coagulative necrosis involving deeper adnexal structures was observed for 75 and 100 J/cm2 light doses. Observed depth of injury increased with increasing radiant exposure, although this relationship was not linear. CONCLUSION: TS-mediated PDT can cause selective vascular injury; however, at higher light doses, significant extra-vascular injury was observed. This information can be used to contribute to design of safe protocols to be used for treatment of cutaneous vascular lesions. Lasers Surg. Med. 49:767-772, 2017. © 2017 Wiley Periodicals, Inc.

Targeted narrowband intense pulsed light on cutaneous vasculature.

BACKGROUND AND OBJECTIVES: Laser based therapies are the standard treatment protocol for port wine stain in the United States, but complete removal is infrequently achieved. Intense pulsed light (IPL) offers a broadband light spectrum approach as a viable treatment alternative. Previous studies suggest that IPL can be more effective in treatment of port wine stain by utilizing multiple wavelengths to selectively target different peaks in oxy- and deoxy-hemoglobin. Our study objectives were to (i) determine a characteristic radiant exposure able to achieve persistent vascular shutdown with narrowband IPL irradiation, (ii) determine the degree to which narrowband IPL irradiation can achieve persistent vascular shutdown, and (iii) compare the effectiveness of narrowband IPL radiation to single wavelength pulsed dye laser (PDL) irradiation in achieving persistent vascular shutdown. STUDY DESIGN/MATERIALS AND METHODS: We utlized either single pulse or double, stacked pulses in narrowband IPL experiments, with the IPL operating over a 500-600 nm wavelength range on the rodent dorsal window chamber model. We compared the results from our narrowband IPL experiments to acquired PDL data from a previous study and determined that narrowband IPL treatments can also produce persistent vascular shutdown. We ran Monte Carlo simulations to investigate the relationship between absorbed energy, wavelength, and penetration depth. RESULTS: For single and double pulse narrowband IPL irradiation we observed (i) little to no change in blood flow, resulting in no persistent vascular shutdown, (ii) marked acute disruption in blood flow and vascular structure, followed by partial to full recovery of blood flow, also resulting in no persistent vascular shutdown, and (iii) immediate changes in blood flow and vascular structure, resulting in prolonged and complete vascular shutdown. Monte Carlo modeling resulted in a 53.2% and 69.0% higher absorbed energy distribution in the top half and the total simulated vessel when comparing the composite narrowband IPL to the 595 nm (PDL), respectively. CONCLUSIONS: Our data collectively demonstrate the potential to achieve removal of vascular lesions using a 500-600 nm range. Additionally, the narrowband IPL was tuned to optimize a specific wavelength range that can be used to treat PWS, whereas the PDL can only operate at one discrete wavelength.