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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. These macrophages 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, such as 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 leads to thrombin-mediated endothelial cell contraction, progressive fibrin accumulation and formation of microthrombi that, once dislodged, cause blockade of vessels in several organs. Together the findings reveal that intravascular resident macrophages are essential to regulate thrombin activity and clear fibrin deposits in regions of disturbed blood flow.

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.

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.

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. Lasers Surg. Med.

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 15–100 J/cm2. We took skin biopsies from subjects at nine 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.

Background: 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. APPROACH: We performed single-cell RNA-sequencing of the ductus arteriosus in mouse embryos at E18.5, and neonatal stages P0.5, and P5 to identify transcriptional alterations that might be associated with remodeling. 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.

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