Mesenteric artery smooth muscle cells from hypertensive rats have increased microtubule acetylation.

The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) - particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the ß-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Proteomic mapping reveals dysregulated angiogenesis in the cerebral arteries of rats with early-onset hypertension.

Hypertension is associated with the presence of vascular abnormalities, including remodeling and rarefaction. These processes play an important role in cerebrovascular disease development; however, the mechanistic changes leading to these diseases are not well characterized. Using data-independent acquisition-based mass spectrometry analysis, here we determined the protein changes in cerebral arteries in pre- and early-onset hypertension from the spontaneously hypertensive rat (SHR), a model that resembles essential hypertension in humans. Our analysis identified 125 proteins with expression levels that were significantly upregulated or downregulated in 12-week-old spontaneously hypertensive rats compared to normotensive Wistar Kyoto rats. Using an angiogenesis enrichment analysis, we further identified a critical imbalance in angiogenic proteins that promoted an anti-angiogenic profile in cerebral arteries at early onset of hypertension. In a comparison to previously published data, we demonstrate that this angiogenic imbalance is not present in mesenteric and renal arteries from age-matched SHRs. Finally, we identified two proteins (Fbln5 and Cdh13), whose expression levels were critically altered in cerebral arteries compared to the other arterial beds. The observation of an angiogenic imbalance in cerebral arteries from the SHR reveals critical protein changes in the cerebrovasculature at the early onset of hypertension and provides novel insights into the early pathology of cerebrovascular disease.

Vascular smooth muscle-specific YAP/TAZ deletion triggers aneurysm development in mouse aorta.

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

Identification of novel proteins and mechanistic pathways associated with early-onset hypertension by deep proteomic mapping of resistance arteries.

Resistance arteries are small blood vessels that create resistance to blood flow. In hypertension, resistance arteries undergo remodeling, affecting their ability to contract and relax appropriately. To date, no study has mapped the hypertension-related proteomic changes in resistance arteries. Using a novel data-independent acquisition-mass spectrometry (DIA-MS) approach, we determined the proteomic changes in small mesenteric and renal arteries in pre- and early-onset hypertension from the spontaneously hypertensive rat (SHR) model, which represents human primary hypertension. Compared with normotensive controls, mesenteric arteries from 12-week-old SHRs had 286 proteins that were significantly up- or downregulated, whereas 52 proteins were identified as up- or downregulated in mesenteric arteries from 6-week-old SHRs. Of these proteins, 18 were also similarly regulated in SHR renal arteries. Our pathway analyses reveal several novel pathways in the pathogenesis of hypertension. Finally, using a matrisome database, we identified 38 altered extracellular-matrix-associated proteins, many of which have never previously been associated with hypertension. Taken together, this study reveals novel proteins and mechanisms that are associated with early-onset hypertension, thereby providing novel insights into disease progression.

Anti-Aß Antibody Aducanumab Regulates the Proteome of Senile Plaques and Closely Surrounding Tissue in a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by accumulation of amyloid-ß (Aß) species and deposition of senile plaques (SPs). Clinical trials with the anti-Aß antibody aducanumab have been completed recently. OBJECTIVE: To characterize the proteomic profile of SPs and surrounding tissue in a mouse model of AD in 10-month-old tgAPPPS1-21 mice after chronic treatment with aducanumab for four months with weekly dosing (10 mg/kg). METHODS: After observing significant reduction of SP numbers in hippocampi of aducanumab-treated mice, we applied a localized proteomic analysis by combining laser microdissection and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the remaining SPs in hippocampi. We microdissected three subregions, containing SPs, SP penumbra level 1, and an additional penumbra level 2 to follow the proteomic profile as gradient. RESULTS: In the aducanumab-treated mice, we identified 17 significantly regulated proteins that were associated with 1) mitochondria and metabolism (ACAT2, ATP5J, ETFA, EXOG, HK1, NDUFA4, NDUFS7, PLCB1, PPP2R4), 2) cytoskeleton and axons (ADD1, CAPZB, DPYSL3, MAG), 3) stress response (HIST1H1C/HIST1H1D, HSPA12A), and 4) AßPP trafficking/processing (CD81, GDI2). These pathways and some of the identified proteins are implicated in AD pathogenesis. Proteins associated with mitochondria and metabolism were mainly upregulated while proteins associated with AßPP trafficking/processing and stress response pathways were mainly downregulated, suggesting that aducanumab could lead to a beneficial proteomic profile around SPs in tgAPPPS1-21 mice. CONCLUSION: We identified novel proteomic patterns of SPs and surrounding tissue indicating that chronic treatment with aducanumab could inhibit Aß toxicity and increase phagocytosis and cell viability.

Proteomic and Unbiased Post-Translational Modification Profiling of Amyloid Plaques and Surrounding Tissue in a Transgenic Mouse Model of Alzheimer's Disease.

Amyloid plaques are one of the hallmarks of Alzheimer's disease (AD). The main constituent of amyloid plaques is amyloid-ß peptides, but a complex interplay of other infiltrating proteins also co-localizes. We hypothesized that proteomic analysis could reveal differences between amyloid plaques and adjacent control tissue in the transgenic mouse model of AD (APPPS1-21) and in similar regions from non-transgenic littermates. Our microproteomic strategy included isolation of regions of interest by laser capture microdissection and analysis by liquid chromatography mass spectrometry-based label-free relative quantification. We consistently identified 183, 224, and 307 proteins from amyloid plaques, adjacent control and non-tg samples, respectively. Pathway analysis revealed 27 proteins that were significantly regulated when comparing amyloid plaques and corresponding adjacent control regions. We further elucidated that co-localized proteins were subjected to post-translational modifications and are the first to report 193 and 117 unique modifications associated to amyloid plaques and adjacent control extracts, respectively. The three most common modifications detected in proteins from the amyloid plaques were oxidation, deamidation, and pyroglutamylation. Together, our data provide novel information about the biological processes occurring within and around amyloid plaques in the APPPS1-21 mouse model of AD.

Dual strategy for reduced signal-suppression effects in matrix-assisted laser desorption/ionization mass spectrometry imaging.

RATIONALE: The molecular complexity of tissue features several signal-suppression effects which reduce the ionization of analytes significantly and thereby weakens the quality of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging (MALDI imaging). We report a novel approach in MALDI imaging by reducing signal-suppression effects for the analysis of beta-amyloid (Aß) plaques, one pathological hallmark of Alzheimer's disease (AD). METHODS: We analyzed Aß proteoforms from postmortem AD brains and brains from transgenic mice (APPPS1-21) overexpressing familial AD mutations by combining two techniques: (1) laser capture microdissection (LCM) to accumulate Aß plaques and (2) phosphoric acid (PA) as additive to the super-2,5-dihydroxybenzoic acid matrix. RESULTS: LCM and MALDI-MS enabled tandem mass spectrometric fragmentation of stained Aß plaques. PA improved the signal-to-noise (S/N) ratio, especially of the Aß1-42 peptide, by three-fold compared with the standard matrix additive trifluoroacetic acid. The beneficial effect of the PA matrix additive in MALDI imaging was particularly important for AD brain tissue. We identified several significant differences in Aß plaque composition from AD compared with APPPS1-21, underlining the value of reducing signal-suppressing effects in MALDI imaging. CONCLUSIONS: We present a novel strategy for overcoming signal-suppression effects in MALDI imaging of Aß proteoforms.

Optimized CLARITY technique detects reduced parvalbumin density in a genetic model of schizophrenia.

BACKGROUND: Novel tissue clearing technologies have, for the first time, made it possible to study intact tissue samples. This approach provides a tool for further clarifying findings from animal models of schizophrenia by studying parvalbumin-positive (PV+) interneuron density from a 3D perspective. NEW METHOD: This study has developed an optimised CLARITY protocol, including an improved electrophoretic tissue clearing (ETC) chamber, an evaluation of antibody diffusion into cleared tissue slices, and a computational method for detecting PV+ interneurons in 3D. RESULTS: A reduced PV+ interneuron density was found in both prelimbic and motor cortex regions of the Df(h15q13)/+ mice, while no changes were observed in the Df(h22q11)/+ mice. COMPARISON WITH EXISTING METHOD: The developed ETC chamber enables tissue clearing of variable tissue sizes while minimizing the resistance. It was found that a high concentration of primary and secondary antibodies were necessary for sufficient antibody staining of PV+ interneurons. Additionally, the developed computational method showed improved detection rates of interneurons compared to non-processed image stacks. CONCLUSION: Our optimization of the CLARITY technology and automated 3D counting of cells were found to be useful for quantification of PV+ interneuron density. The results may provide insight into understanding the pathophysiology underlying the phenotype observed in Df(h15q13)/+ mice.