Chimeric antigen receptor macrophages target and resorb amyloid plaques.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer's disease (AD). While the precise pathophysiology of AD is incompletely understood, clinical trials of antibodies targeting aggregated forms of ß amyloid (Aß) have shown that reducing amyloid plaques can mitigate cognitive decline in patients with early-stage AD. Here, we describe what we believe to be a novel approach to target and degrade amyloid plaques by genetically engineering macrophages to express an Aß-targeting chimeric antigen receptor (CAR-Ms). When injected intrahippocampally, first-generation CAR-Ms have limited persistence and fail to significantly reduce plaque load, which led us to engineer next-generation CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. Cytokine secreting "reinforced CAR-Ms" have greater survival in the brain niche and significantly reduce plaque load locally in vivo. These findings support CAR-Ms as a platform to rationally target, resorb, and degrade pathogenic material that accumulates with age, as exemplified by targeting Aß in AD.

Holotype sequencing of Silvataresholzenthali Rázuri-Gonzales, Ngera & Pauls, 2022 (Trichoptera, Pisuliidae).

While DNA barcodes are increasingly provided in descriptions of new species, the whole mitochondrial and nuclear genomes are still rarely included. This is unfortunate because whole genome sequencing of holotypes allows perpetual genetic characterization of the most representative specimen for a given species. Thus, de novo genomes are invaluable additional diagnostic characters in species descriptions, provided the structural integrity of the holotype specimens remains intact. Here, we used a minimally invasive method to extract DNA of the type specimen of the recently described caddisfly species Silvataresholzenthali Rázuri-Gonzales, Ngera & Pauls, 2022 (Trichoptera: Pisuliidae) from the Democratic Republic of the Congo. A low-cost next generation sequencing strategy was used to generate the complete mitochondrial and draft nuclear genome of the holotype. The data in its current form is an important extension to the morphological species description and valuable for phylogenomic studies.

Astrocytic APOE4 removal confers cerebrovascular protection despite increased cerebral amyloid angiopathy.

BACKGROUND: Alzheimer Disease (AD) and cerebral amyloid angiopathy (CAA) are both characterized by amyloid-ß (Aß) accumulation in the brain, although Aß deposits mostly in the brain parenchyma in AD and in the cerebrovasculature in CAA. The presence of CAA can exacerbate clinical outcomes of AD patients by promoting spontaneous intracerebral hemorrhage and ischemia leading to CAA-associated cognitive decline. Genetically, AD and CAA share the ε4 allele of the apolipoprotein E (APOE) gene as the strongest genetic risk factor. Although tremendous efforts have focused on uncovering the role of APOE4 on parenchymal plaque pathogenesis in AD, mechanistic studies investigating the role of APOE4 on CAA are still lacking. Here, we addressed whether abolishing APOE4 generated by astrocytes, the major producers of APOE, is sufficient to ameliorate CAA and CAA-associated vessel damage. METHODS: We generated transgenic mice that deposited both CAA and plaques in which APOE4 expression can be selectively suppressed in astrocytes. At 2-months-of-age, a timepoint preceding CAA and plaque formation, APOE4 was removed from astrocytes of 5XFAD APOE4 knock-in mice. Mice were assessed at 10-months-of-age for Aß plaque and CAA pathology, gliosis, and vascular integrity. RESULTS: Reducing the levels of APOE4 in astrocytes shifted the deposition of fibrillar Aß from the brain parenchyma to the cerebrovasculature. However, despite increased CAA, astrocytic APOE4 removal reduced overall Aß-mediated gliosis and also led to increased cerebrovascular integrity and function in vessels containing CAA. CONCLUSION: In a mouse model of CAA, the reduction of  APOE4 derived specifically from astrocytes, despite increased fibrillar Aß deposition in the vasculature, is sufficient to reduce Aß-mediated gliosis and cerebrovascular dysfunction.

Elevational biodiversity gradients in the Neotropics: Perspectives from freshwater caddisflies (Insecta: Trichoptera).

Aquatic insects in the order Trichoptera are extremely diverse in number of species and their trophic roles. However, their distribution and diversity patterns are poorly known in the Neotropics, including the species restricted to tropical mountain ecosystems. Recent studies in tropical mountains have shown high levels of endemism of aquatic insects and changes in the composition of communities over short distances. Still, the incidence of environmental filters that explain such patterns has not been addressed quantitatively. Given the relevance of understanding Trichoptera spatial diversity patterns to prioritize conservation areas for freshwaters, as well as to obtain baseline information to predict changes in aquatic communities facing global environmental changes, we assessed the species distribution and assemblages of caddisflies along an elevational gradient from 600 to 3,600 m a.s.l. on the equatorial Andes. In this area, we had long-term continuous climate data with hourly resolution. We collected adult caddisflies in seven localities along this gradient using light traps. We sampled each locality for two hours after sunset for three consecutive days. All specimens collected were identified to species or morphospecies. Our results showed an increase in species and genera numbers with decreasing altitude, albeit no significant. Minimum air temperature is the main environmental variable explaining Trichoptera community assemblages. ß-diversity (taxon turnover among sites), as opposed to species richness, increased with altitude and showed a bimodal distribution along the elevation gradient for both genera and species assemblages, which resulted in a significant shift in community composition of species and genera at 2,000 m a.s.l. Our null-models confirm the observed patterns of B-diversity are non-random and suggest a strong environmental filtering of tropical caddisflies community assemblies and turnover. Geographic distance coupled with changes in environmental conditions along the elevation gradient explained a high percentage of community variance, as documented for other taxa (e.g., vascular plants), suggesting the importance of securing habitat connectivity along the altitudinal gradient to protect aquatic insect diversity effectively.

De Novo Genome Assembly and Annotation of an Andean Caddisfly, Atopsyche davidsoni Sykora, 1991, a Model for Genome Research of High-Elevation Adaptations.

We sequence, assemble, and annotate the genome of Atopsyche davidsoni Sykora, 1991, the first whole-genome assembly for the caddisfly family Hydrobiosidae. This free-living and predatory caddisfly inhabits streams in the high-elevation Andes and is separated by more than 200 Myr of evolutionary history from the most closely related caddisfly species with genome assemblies available. We demonstrate the promise of PacBio HiFi reads by assembling the most contiguous caddisfly genome assembly to date with a contig N50 of 14 Mb, which is more than 6× more contiguous than the current most contiguous assembly for a caddisfly (Hydropsyche tenuis). We recover 98.8% of insect BUSCO genes indicating a high level of gene completeness. We also provide a genome annotation of 12,232 annotated proteins. This new genome assembly provides an important new resource for studying genomic adaptation of aquatic insects to harsh, high-altitude environments.

A new species of Silvatares (Trichoptera, Pisuliidae) from the Democratic Republic of the Congo.

A new species of caddisfly in the family Pisuliidae from the Democratic Republic of the Congo is described and illustrated herein, Silvataresholzenthali sp. nov. Based on the presence of a pair of spines on the endotheca, this species belongs to the thrymmifer group. Additionally, Silvatareslaetae is recorded for the first time from the D.R. Congo.

APOE immunotherapy reduces cerebral amyloid angiopathy and amyloid plaques while improving cerebrovascular function.

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and greatly influences the development of amyloid-ß (Aß) pathology. Our current study investigated the potential therapeutic effects of the anti-human APOE antibody HAE-4, which selectively recognizes human APOE that is co-deposited with Aß in cerebral amyloid angiopathy (CAA) and parenchymal amyloid pathology. In addition, we tested whether HAE-4 provoked brain hemorrhages, a component of amyloid-related imaging abnormalities (ARIA). ARIA is an adverse effect secondary to treatment with anti-Aß antibodies that can occur in blood vessels with CAA. We used 5XFAD mice expressing human APOE4 +/+ (5XE4) that have prominent CAA and parenchymal plaque pathology to assess the efficacy of HAE-4 compared to an Aß antibody that removes parenchymal Aß but increases ARIA in humans. In chronically treated 5XE4 mice, HAE-4 reduced Aß deposition including CAA compared to a control antibody, whereas the anti-Aß antibody had no effect on CAA. Furthermore, the anti-Aß antibody exacerbated microhemorrhage severity, which highly correlated with reactive astrocytes surrounding CAA. In contrast, HAE-4 did not stimulate microhemorrhages and instead rescued CAA-induced cerebrovascular dysfunction in leptomeningeal arteries in vivo. HAE-4 not only reduced amyloid but also dampened reactive microglial, astrocytic, and proinflammatory-associated genes in the cortex. These results suggest that targeting APOE in the core of both CAA and plaques could ameliorate amyloid pathology while protecting cerebrovascular integrity and function.

Reduction of Leukocyte Microvascular Adherence and Preservation of Blood-Brain Barrier Function by Superoxide-Lowering Therapies in a Piglet Model of Neonatal Asphyxia.

Background: Asphyxia is the most common cause of brain damage in newborns. Substantial evidence indicates that leukocyte recruitment in the cerebral vasculature during asphyxia contributes to this damage. We tested the hypothesis that superoxide radical ( O 2 ⋅ _ ) promotes an acute post-asphyxial inflammatory response and blood-brain barrier (BBB) breakdown. We investigated the effects of removing O 2 ⋅ _ by superoxide dismutase (SOD) or C3, the cell-permeable SOD mimetic, in protecting against asphyxia-related leukocyte recruitment. We also tested the hypothesis that xanthine oxidase activity is one source of this radical. Methods: Anesthetized piglets were tracheostomized, ventilated, and equipped with closed cranial windows for the assessment of post-asphyxial rhodamine 6G-labeled leukocyte-endothelial adherence and microvascular permeability to sodium fluorescein in cortical venules. Asphyxia was induced by discontinuing ventilation. SOD and C3 were administered by cortical superfusion. The xanthine oxidase inhibitor oxypurinol was administered intravenously. Results: Leukocyte-venular adherence significantly increased during the initial 2 h of post-asphyxial reperfusion. BBB permeability was also elevated relative to non-asphyxial controls. Inhibition of O 2 ⋅ _ production by oxypurinol, or elimination of O 2 ⋅ _ by SOD or C3, significantly reduced rhodamine 6G-labeled leukocyte-endothelial adherence and improved BBB integrity, as measured by sodium fluorescein leak from cerebral microvessels. Conclusion: Using three different strategies to either prevent formation or enhance elimination of O 2 ⋅ _ during the post-asphyxial period, we saw both reduced leukocyte adherence and preserved BBB function with treatment. These findings suggest that agents which lower O 2 ⋅ _ in brain may be attractive new therapeutic interventions for the protection of the neonatal brain following asphyxia.

New Atanatolica species from Ecuador (Trichoptera, Leptoceridae).

Four new species of Atanatolica Mosely are described from Ecuador: A.andina sp. n., A.angulata sp. n., A.curvata sp. n., and A.decouxi sp. n. These species belong to the A.dominicana group and constitute new records of the genus from Chimborazo, Imbabura, and Napo Provinces. Additionally, A.andina sp. n. represents the highest elevation recorded for any species in the genus at 3900 m. Size class data are also presented suggesting continuous larval growth for the probable larva of A.decouxi sp. n., described and illustrated here. A new distribution record is provided for A.manabi from Carchi Province.

Two new species of the rare Neotropical caddisfly genus Amphoropsyche Holzenthal (Trichoptera, Leptoceridae).

Two new species in the rare, endemic Neotropical caddisfly genus Amphoropsyche Holzenthal, 1985 are described from Ecuador (A. carchisp. n.) and Peru (A. matsigenkasp. n.) bringing to 17 the number of species known in the genus. Almost all species are known from only a few individuals and from even fewer localities. The new species belong to a group of 10 other species that have tergum X in the male genitalia divided into a mesal process and a pair of lateral processes. Amphoropsyche carchi can be separated from those species by the rounded mesal concavity, the short mesobasal lobe, and the short 2nd article of the inferior appendage, while A. matsigenka can be diagnosed by the very slender and straight inferior appendage, which bears a pair of spine-like mesoventral projections. We also present a new record for Amphoropsyche tandayapa Holzenthal & Rázuri-Gonzales, 2011, from Ecuador, previously known only from the male holotype.

New species of the endemic Neotropical caddisfly genus Contulma from the Andes of Ecuador (Trichoptera: Anomalopsychidae).

The genus Contulma Flint (Trichoptera: Anomalopsychidae) is composed mostly of regionally endemic species occurring above 2,000 m, with a few more widespread species and some that are found at lower elevations. Adults of three new species of Contulma are described and illustrated from the Andes of Ecuador, Contulma lina, new species, Contulma quito, new species, and Contulma sangay, new species. These species are similar to previously described species from the region, including C. paluguillensis, C. nevada, and C. lancelolata. New provincial records are provided for C. bacula, C. cataracta, and C. echinata. Contulma duffi Oláh, 2016 is considered a junior, subjective synonym of C. penai, Holzenthal & Flint, 1995. Also, we provide an identification key to males of the 30 Contulma species now known.

Diversity and distribution of the Caddisflies (Insecta: Trichoptera) of Ecuador.

BACKGROUND: Aquatic insects and other freshwater animals are some of the most threatened forms of life on Earth. Caddisflies (Trichoptera) are highly biodiverse in the Neotropics and occupy a wide variety of freshwater habitats. In Andean countries, including Ecuador, knowledge of the aquatic biota is limited, and there is a great need for baseline data on the species found in these countries. Here we present the first list of Trichoptera known from Ecuador, a country that harbors two global biodiversity "hotspots." METHODS: We conducted a literature review of species previously reported from Ecuador and supplemented these data with material we collected during five recent field inventories from about 40 localities spanning both hotspots. Using species presence data for each Ecuadorian province, we calculated the CHAO 2 species estimator to obtain the minimum species richness for the country. RESULTS: We recorded 310 species, including 48 new records from our own field inventories for the country. CHAO 2 calculations showed that only 54% of the species have been found. Hydroptilidae and Hydropsychidae were the most species rich families. We report the family Xiphocentronidae for the first time from Ecuador as well as several new records of genera from different families. DISCUSSION: As in the neighboring Andean countries of Colombia and Peru, it is common to find undescribed species of caddisflies. There are vast areas of Ecuador and the northern Andes that are completely unexplored, and we expect that hundreds of new species are yet to be discovered.

New synonyms in the highly diverse caddisfly genus Smicridea (Trichoptera, Hydropsychidae).

In this paper, Smicridea (Rhyacophylax) repula Oláh & Johanson, 2012 is synonymized with Smicridea (Rhyacophylax) lobata (Ulmer, 1909), and the species Leptonema islamarga Botosaneanu, 2002 is transferred to Smicridea (Rhyacophylax) as a synonym of Smicridea lobata. Additionally, we present more detailed illustrations of the male genitalia of Smicridea (Rhyacophylax) lobata and Smicridea (Rhyacophylax) signata (Banks, 1903), and include notes on their distributions to aid in the identification of these two, often-confused species.

Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aß Generation and Amyloid Plaque Pathogenesis.

In AD, an imbalance between Aß production and removal drives elevated brain Aß levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via α-cleavage at the plasma membrane, amyloidogenic ß- and γ-cleavage within endosomes to generate Aß, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aß levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adeno-associated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the α and ß C-terminal APP fragments (CTFs), and in the steady-state Aß levels in the brain interstitial fluid. In aged mice, total Aß levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and ß-CTFs, and attenuated Aß generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aß generation. Activation of TFEB in neurons is an effective strategy to attenuate Aß generation and attenuate amyloid plaque deposition in AD. SIGNIFICANCE STATEMENT: A key driver for AD pathogenesis is the net balance between production and clearance of Aß, the major component of amyloid plaques. Here we demonstrate that lysosomal degradation of holo-APP influences Aß production by limiting the availability of APP for amyloidogenic processing. Using viral gene transfer of transcription factor EB (TFEB), a master regulator of lysosome biogenesis in neurons of APP/PS1 mice, steady-state levels of APP were reduced, resulting in decreased interstitial fluid Aß levels and attenuated amyloid deposits. These effects were caused by accelerated lysosomal degradation of endocytosed APP, reflected by reduced APP half-life and steady-state levels in TFEB-expressing cells, with resultant decrease in Aß production and release. Additional studies are needed to explore the therapeutic potential of this approach.

Enhancing astrocytic lysosome biogenesis facilitates Aß clearance and attenuates amyloid plaque pathogenesis.

In sporadic Alzheimer's disease (AD), impaired Aß removal contributes to elevated extracellular Aß levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood-brain barrier, and cellular uptake facilitate physiologic Aß clearance. Astrocytes can take up and degrade Aß, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate Aß removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein Aß localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote Aß uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied Aß42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of Aß. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced Aß levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate Aß removal and counter amyloid plaque pathogenesis in AD.

Bcl-x pre-mRNA splicing regulates brain injury after neonatal hypoxia-ischemia.

The bcl-x gene appears to play a critical role in regulating apoptosis in the developing and mature CNS and following CNS injury. Two isoforms of Bcl-x are produced as a result of alternative pre-mRNA splicing: Bcl-x(L) (the long form) is anti-apoptotic, while Bcl-x(S) (short form) is pro-apoptotic. Despite the antagonistic activities of these two isoforms, little is known about how regulation of alternative splicing of bcl-x may mediate neural cell apoptosis. Here, we report that apoptotic stimuli (staurosporine or C2-ceramide) reciprocally altered Bcl-x splicing in neural cells, decreasing Bcl-x(L) while increasing Bcl-x(S). Specific knockdown of Bcl-x(S) attenuated apoptosis. To further define regulatory elements that influenced Bcl-x splicing, a Bcl-x minigene was constructed. Deletional analysis revealed several consensus sequences within intron 2 that altered splicing. We found that the splicing factor, CUG-binding-protein-1 (CUGBP1), bound to a consensus sequence close to the Bcl-x(L) 5' splice site, altering the Bcl-x(L)/Bcl-x(S) ratio and influencing cell death. In vivo, neonatal hypoxia-ischemia reciprocally altered Bcl-x pre-mRNA splicing, similar to the in vitro studies. Manipulation of the splice isoforms using viral gene transfer of Bcl-x(S) shRNA into the hippocampus of rats before neonatal hypoxia-ischemia decreased vulnerability to injury. Moreover, alterations in nuclear CUGBP1 preceded Bcl-x splicing changes. These results suggest that alternative pre-mRNA splicing may be an important regulatory mechanism for cell death after acute neurological injury and may potentially provide novel targets for intervention.

Dysmyelinated axons in shiverer mice are highly vulnerable to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated toxicity.

Glutamate excitotoxicity plays a role in white matter injury in many neurological diseases. Oligodendrocytes in particular are highly vulnerable to excitotoxicity, mediated through activation of AMPA/kainate receptors. Myelin may also be injured independently via NMDA (N-methyl-D-aspartic acid) receptors located on peripheral oligodendroglial processes. Central axons are susceptible to glutamate receptor activation in vivo, but it is unclear whether this is mediated directly by activation of receptors expressed on axons, or indirectly through glutamate toxicity of myelin or neighboring glial cells. We examined axonal vulnerability in mice deficient in myelin basic protein (shiverer), also expressing yellow fluorescent protein (YFP) in a subset of axons. YFP fluorescence, EM, and mouse behavior were assessed 24 h after microstereotactical injections of S-AMPA or NMDA into lumbar dorsal columns. S-AMPA injection led to impaired rotarod performance and widespread axonal degeneration and was more pronounced in shiverer mice than controls. In contrast, NMDA injection did not cause axonal injury or behavioral changes in either group. These results indicate that spinal cord axons in vivo are vulnerable to toxicity mediated by AMPA but not NMDA receptors. The presence of compact myelin is not required for excitotoxic axon damage, and its absence may increase vulnerability. Further understanding of AMPA receptor-mediated axonal toxicity may provide new targets for neuroprotective therapy in WM diseases.

Characterizing the appearance and growth of amyloid plaques in APP/PS1 mice.

Amyloid plaques are primarily composed of extracellular aggregates of amyloid-beta (Abeta) peptide and are a pathological signature of Alzheimer's disease. However, the factors that influence the dynamics of amyloid plaque formation and growth in vivo are largely unknown. Using serial intravital multiphoton microscopy through a thinned-skull cranial window in APP/PS1 transgenic mice, we found that amyloid plaques appear and grow over a period of weeks before reaching a mature size. Growth was more prominent early after initial plaque formation: plaques grew faster in 6-month-old compared with 10-month-old mice. Plaque growth rate was also size-related, as smaller plaques exhibited more rapid growth relative to larger plaques. Alterations in interstitial Abeta concentrations were associated with changes in plaque growth. Parallel studies using multiphoton microscopy and in vivo microdialysis revealed that pharmacological reduction of soluble extracellular Abeta by as little as 20-25% was associated with a dramatic decrease in plaque formation and growth. Furthermore, this small reduction in Abeta synthesis was sufficient to reduce amyloid plaque load in 6-month-old but not 10-month-old mice, suggesting that treatment early in disease pathogenesis may be more effective than later treatment. In contrast to thinned-skull windows, no significant plaque growth was observed under open-skull windows, which demonstrated extensive microglial and astrocytic activation. Together, these findings indicate that individual amyloid plaque growth in vivo occurs over a period of weeks and may be influenced by interstitial Abeta concentration as well as reactive gliosis.

Neutrophil elastase and neurovascular injury following focal stroke and reperfusion.

Neutrophil elastase (NE) degrades basal lamina and extracellular matrix molecules, and recruits leukocytes during inflammation; however, a basic understanding of the role of NE in stroke pathology is lacking. We measured an increased number of extravascular NE-positive cells, as well as increased levels of tissue elastase protein and activity, following transient middle cerebral artery occlusion (tMCAo). Both pharmacologic inhibition of NE with ZN200355 (ZN), and genetic deletion of NE, significantly reduced infarct volume, blood-brain barrier disruption, vasogenic edema, and leukocyte-endothelial adherence 24 h after tMCAo. ZN also reduced infarct volume in MMP9-null mice following tMCAo. There were, however, no reductions in infarct volume or vasogenic edema in NE-null mice in two models of permanent middle cerebral artery occlusion. Our findings confirm the involvement of NE in neurovascular stroke pathology, when reperfusion allows neutrophils access to vulnerable brain, with pharmacologic or genetic inhibition of NE being both neuro- and vasculo-protective in this setting.