An update on advances in magnetic resonance imaging of multiple system atrophy

In this review, we describe how different neuroimaging tools have been used to identify novel MSA biomarkers, highlighting their advantages and limitations. First, we describe the main structural MRI changes frequently associated with MSA including the ‘hot cross-bun’ and ‘putaminal rim’ signs as well as putaminal, pontine, and middle cerebellar peduncle (MCP) atrophy. We discuss the sensitivity and specificity of different supra- and infratentorial changes in differentiating MSA from other disorders, highlighting those that can improve diagnostic accuracy, including the MCP width and MCP/superior cerebellar peduncle (SCP) ratio on T1-weighted imaging, raised putaminal diffusivity on diffusion-weighted imaging, and increased T2* signal in the putamen, striatum, and substantia nigra on susceptibility-weighted imaging. Second, we focus on recent advances in structural and functional MRI techniques including diffusion tensor imaging (DTI), resting-state functional MRI (fMRI), and arterial spin labelling (ASL) imaging. Finally, we discuss new approaches for MSA research such as multimodal neuroimaging strategies and how such markers may be applied in clinical trials to provide crucial data for accurately selecting patients and to act as secondary outcome measures.

Introduction
Multiple system atrophy (MSA) is an adult-onset, neurodegenerative disorder characterised by parkinsonism, ataxia, and dysautonomia. The neuropathological hallmark is alphasynuclein-positive glial cytoplasmic inclusions (GCIs) with degeneration of the striatal, nigral, and olivopontine structures. The deposition of alpha-synuclein links MSA with other synucleinopathies, including idiopathic Parkinson's disease (IPD) and dementia with Lewy bodies (DLB). However, clinically, there is more commonly overlap with atypical parkinsonian syndromes such as progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS).
Diagnostic accuracy in multiple system atrophy (MSA) varies greatly between different centres from as little as 29% up to 86% [1][2][3][4], despite well-established diagnostic criteria [5]. While a definite MSA diagnosis can only be reached with post-mortem confirmation of GCIs in a well-defined pattern, a probable MSA diagnosis is considered when a poor levodopa-responsive parkinsonian syndrome (MSA-P) and/or a cerebellar syndrome (MSA-C) is associated with autonomic failure. A possible MSA diagnosis is defined when sporadic adult-onset parkinsonism (MSA-P), or a cerebellar syndrome (MSA-C) is accompanied by autonomic dysfunction and at least one item from a list of additional red-flag features [5,6]. These include structural and functional neuroimaging changes that have become established over the last two decades as useful diagnostic markers. However, while such abnormalities are helpful diagnostically, brain-imaging research in MSA has expanded to examine a variety of post-processing techniques and more advanced imaging modalities that may potentially lead to much improved biomarkers for diagnosis and disease progression. In this review, we describe how different neuroimaging tools have been used to identify novel MSA biomarkers, highlighting their advantages and limitations.

Structural MR imaging
Historically, most MSA neuroimaging research has been focused on the grey matter atrophy pattern seen on structural T1 MRI and signal changes seen on T2, FLAIR, and T2* MRI. The main aims of these studies have been to: (a) provide a diagnostic MSA atrophy pattern useful for consensus criteria; (b) distinguish between the two clinical forms MSA-P and MSA-C; and (c) improve the differential diagnosis with other neurodegenerative conditions that mimic MSA, especially with IPD, DLB, PSP, and CBS.
Visualisation of T1-and T2-weighted MRI by experienced neuroradiologists has been the cornerstone of MSA-imaging diagnosis for many years, and a number of 'classical' signs have been described. The 'hot cross-bun' sign is the most well known (Fig. 1a) and represents the degeneration of the pons and pontocerebellar fibres with the preservation of corticospinal tract. It appears as a hyperintense cross in the pons on T2-weighted imaging. Despite being a hallmark for MSA-C with high specificity (97%), its sensitivity is only 50% [7]. The 'putaminal rim' sign is another well-described imaging feature of MSA that has acquired its own name (Fig. 1b). The presence of a hyperintense rim to the putamen on T2*-weighted imaging is seen in MSA-P and has the highest specificity (90%) for this clinical subgroup but only 72% sensitivity [8]. It can also be a normal finding on 3T T2-weighted MRI.
Other putaminal changes described in MSA-P include atrophy (seen on T1) (sensitivity 83%, specificity 87%), and hypointensity (sensitivity 89%, specificity 70%- Fig. 1b) [8], while in MSA-C, there are changes infratentorially with hyperintensity of the middle cerebellar peduncle (MCP) and atrophy of the cerebellum and brainstem (particularly the MCP and pons) [10] (sensitivity 100%, specificity 82% for brainstem atrophy) [11]. In fact, a combination of these signs can be seen in the majority of MSA patients at different stages of the disease, independent of the initial clinical phenotype [8,12].
While none of these signs are pathognomonic, their presence has been shown to have high specificity but lower sensitivity in differentiating MSA from other disorders [13], e.g., the presence of MCP hyperintensity has a specificity of 100% and sensitivity of 85% when compared with IPD, PSP, and normal aging controls [14]. Fig. 1 a ''Hot cross-bun sign'' seen on an axial T2-weighted MRI in a patient with MSA-C; b putaminal hypointensity with a hyperintense "putaminal rim" sign on an axial T2-weighted MRI in a patient with MSA-P. Images adapted from [9] Simply applied quantitative measures have been shown to improve diagnostic accuracy. A study that measured MCP atrophy as a reduction of the MCP width (< 8 mm in sagittal sections) showed 100% sensitivity and specificity in differentiating MSA from IPD patients [15,16], while another study investigating the MCP/superior cerebellar peduncle (SCP) ratio had a sensitivity of 90% and specificity of 94% when comparing MSA-P to PSP [17]. Adding in measurements of the pons and midbrain allowed one study to define an MR parkinsonism index [= pontine area/midbrain area)*(MCP/SCP)] which differentiated MSA-P from PSP and IPD with high sensitivity and specificity [17].
More detailed quantitative methods of assessing crosssectional grey matter atrophy analyses have been applied in research settings. These have usually consisted of either Region-of-Interest (ROI) volumetric analyses (performed either manually or in a more automated manner), or whole brain analyses such as voxel-based morphometry (VBM) [18].
ROI studies using semiautomatic segmentation and MRI volumetry (MRV) techniques showed a combination of supra-and infratentorial volume loss including striatum, brainstem, and cerebellum in MSA [19,20]. A more accurate differentiation between MSA and other parkinsonian syndromes was achieved with the application of a stepwise discriminant analysis [11,19]. Volume loss in the basal ganglia and infratentorial brain regions have been confirmed by VBM studies [21][22][23][24]. VBM is an automated method of measuring neuroanatomical changes in the grey matter using 3D volumetric T1-weighted MR imaging. Compared to controls, selective cortical atrophy involving the primary and higher order motor areas, prefrontal cortex, and insula was identified in MSA-P cases [21] (Fig. 2) and confirmed on a longitudinal VBM study [22]. A meta-analysis assessed the use of VBM in differentiating MSA-P, IPD, and normal Images are in the neuroradiological orientation (the left side of the images refers to the right side of the brain). VBM in MSA-P a grey matter loss, b correlation of grey matter loss with disease stage, and c increase of white matter. VBM and VBR compari-son between MSA-C and MSA-P: the images display regions with more pronounced changes in MSA-C than in MSA-P. a Grey matter loss, b white matter loss, and c reduced relaxation rate. Images reproduced from [26] controls [25]: although a different pattern and localization of grey matter reduction was identified in the MSA-P versus IPD group (atrophy in the putamen and claustrum), the differences were not significant in a subgroup analysis including only patients in early stages with a mean disease duration of less than three years.
Longitudinal analyses of atrophy rates are limited (Fig. 3). These studies are important, as the atrophy rate is an important quantitative marker of disease progression that has been successfully implemented in other neurodegenerative conditions as an outcome measure in clinical trials [27][28][29]. Only two studies have assessed whole brain atrophy rate (WBAR) in MSA [30,31]. Although these had a short follow-up and assessed a small number of cases, they showed that the WBAR was higher from the early stages of MSA (and PSP) compared with IPD, suggesting that this could be used as an unbiased outcome measure for monitoring the disease course in future clinical trials. In addition, as MSA is a rapidly progressive disorder, using imaging measurements improves reliability compared to clinical disease rating scores [11,19,30].

DWI
Diffusion-weighted imaging (DWI) uses water molecule movement and calculates the apparent diffusion coefficient (ADC) in tissue as a measure of integrity. In neurodegeneration and ischemia, the random movement of water molecules is increased. One of the most promising DWI markers for MSA is raised putaminal diffusivity in MSA-P compared to PD, even in early stages of disease [33][34][35][36] (Fig. 4). DWI was also helpful in differentiating MSA-P from PSP where increased regional ADC in the MCP and pons in MSA-P, compared to PSP, had 91% sensitivity and 84% specificity Delineating the sites and progression of in vivo atrophy in multiple system atrophy using fluid-registered MRI. Coronal MRI scan with voxel-compression-mapping overlay to demonstrate areas undergoing atrophy. Greatest rates of atrophy are demonstrated in the pons and middle cerebellar peduncles and the immediately adjacent midbrain and medulla. Increased atrophy, but at a slower rate, is seen in the upper midbrain and lower medulla. Even slower, but definitely pathological atrophy rates are seen in both temporal lobes. Ventricular enlargement is also shown. Image reproduced from [32] , c). The PD patient shows no increased Trace (D) values in the putamen, neither at baseline (e) nor at follow-up (f). Images reproduced from [39] [37]. DWI-measured progression of striatal and extrastriatal degeneration including the putamen, pons, and cerebellar white matter in MSA in a longitudinal study correlated well with disease duration and severity at 1-year follow-up [38].

MTR
Magnetization transfer imaging allows brain structure segmentation and ratio (MTR) calculation of a specific ROI. A significant decrease in MTR of the globus pallidus, putamen, and substantia nigra has been reported in MSA compared to IPD [40]. Changes in the basal ganglia were reported in MSA-P in studies using MTR [40,41]. Furthermore, using basal ganglia and substantia nigra changes in stepwise MRT analysis provided a good discrimination rate between PD cases and controls from the MSA and PSP group. However, the classification of individual MSA and PSP cases into disease groups was not optimal [40].

Susceptibility-weighted imaging (SWI) is a gradient echo
image that provides information about any tissue that has a different magnetic susceptibility compared to its surrounding structures such as deoxygenated blood, hemosiderin, ferritin, and calcium [42]. Compared with a standard T2* sequence, there is increased sensitivity in detecting local changes in iron content [43]. This is important in MSA, as several histopathological studies have revealed increased iron and ferritin levels in the putamen (particularly posteriorly), striatum and substantia nigra [44], and at a significantly higher amount than in IPD [45]. SWI studies have shown a much higher iron deposition in the putamen and pallidum of MSA-P compared to IPD and PSP [46,47]. One study splitting the putamen into four regions suggested that the lower inner part is the best marker to differentiate between MSA-P and IPD [48]. Another study showed increasing iron accumulation in the putamen from posterolateral areas in the early stages to more anteromedial areas later [49]. Studies of the caudate are less clear: one study found an increased deposition of iron in the caudate nucleus of MSA-P compared to IPD [48], while a second study could not replicate these findings based on SWI alone [49]. These contradictory results may well represent differences in disease duration in the different sample groups.

VBR
Voxel-based relaxometry (VBR) is a morphometric method that analyses the relaxation rate R2 (defined as 1/T2) derived from multi-echo T2-weighted images on a voxel by-voxel basis using the exponential relationship between the actual transverse magnetization and the relaxation rate R2. In principle, decreased R2 indicates increased water content and, therefore, provides a measure of tissue atrophy [26]. VBR analysis in MSA-C patients revealed a reduced relaxation rate R2 particularly within the cerebellum, middle cerebellar peduncles, and pons [19,24,50,51].

QSM
Quantitative susceptibility mapping (QSM) detects local susceptibility changes to metals such as iron. Studies suggested that QSM is a better tool for measuring iron levels in the tissue [52,53]. Both the R2 and QSM are increased in MSA and PSP compared to PD and controls in several brain structures including the basal ganglia and cerebellum. However, compared to PSP, the MSA subgroup had different iron deposition patterns in the SN, thalamus, and red nucleus [54,55]. A post-mortem study assessed the QSM and R2 in path confirmed parkinsonian disorders showing that in vivo increased R2 was significantly associated with alpha-synuclein and QSM correlated significantly with Perl's stain for iron. However, neither measurement correlated with tau nor glial cell counts [53].

Structural and functional connectivity
Diffusion tensor imaging (DTI) uses the motion of water to quantify changes in the microstructure of white matter tracts [56]. DTI metrics commonly include fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD). FA is a marker of fibre structural integrity, being reduced when neuronal fibres are destroyed causing water diffusion to occur in all directions (become isotropic). Diffusivity measures are higher [57], with AD considered a reflection of axonal loss and RD of myelin damage [58].
Several studies have used DTI to assess white matter tract changes in MSA. DTI shows that the cerebellum, in particular, MCP regions, and globus pallidum of MSA patients have reduced FA associated with higher MD values, compared to IPD [59,60] (Fig. 5).
One study compared DTI in different MSA subtypes using the Diffusion Trace (D) generating brain maps from the MD images in the three orthogonal directions. Trace (D) values measured in the entire and anterior putamen were significantly higher in MSA-P than in MSA-C cases, whereas Trace (D) values in the cerebellum and middle cerebellar peduncle (MCP) were significantly higher in MSA-C than in MSA-P patients and controls. Furthermore The hot cross-bun sign implies that the corticospinal tract (CST) remains intact in the presence of degeneration of pons and pontocerebellar fibres. However, this contradicts pathological reports that support the involvement of white matter CST in MSA. Using DTI CST, white matter changes have been shown to be present, with a marked decrease in FA and increase in MD observed in the transverse pontocerebellar fibres, the corticospinal tracts, pons, and the cerebellum. These observations correlate well with neuropathological studies [62], suggesting that DTI is much better at detecting white matter structural changes than the standard MRI sequences. Resting-state functional MRI (rsfMRI) is a relatively new tool that, unlike task-based fMRI, assesses brain connectivity, while the subject is at 'rest' (lying quietly in the scanner). This technique allows the assessment of 'functional' connectivity and networks by visualising synchronised neuronal activation (based on the blood oxygenation level or BOLD signal) between spatially distinct brain regions [63]. The most well-defined network is the default mode network [64], although multiple other 'intrinsic connectivity networks' have been described including sensorimotor, visual, language, attention, and salience networks [65][66][67][68][69][70]. There are currently only a small number of studies using rsfMRI in MSA. The first such study showed that the networks most affected in MSA are the default mode and sensorimotor networks [71]. 20 clinically probable MSA patients were analysed alongside 9 healthy controls, using a regional homogeneity (ReHo) method to investigate neuronal networks in resting state. ReHo changes have been described in IPD, including sensorimotor networks [72], but in the IPD group, ReHo was reduced in medial PFC and SMA, compared with an increase in the same areas in MSA. Although these are very small studies and require further replication, they could be significant. However, other pathways affected in MSA, e.g., the olivopontocerebellar and the nigrostriatal networks, were not assessed, so much remains to be explored. Whole brain connectivity analysis was also used to monitor response to repetitive transcranial magnetic stimulation (TMS) in MSA patients in a small study [73]. Patients were randomised to 10 sessions of TMS targeting the motor cortex area or sham TMS. Patients receiving active TMS showed changes in several networks including the default mode, cerebellar, and limbic networks. Interestingly, the positive changes in the functional networks were associated with improved motor symptoms in the TMS-treated group [73].

Measures of perfusion
Arterial spin labelling (ASL) is a new MRI technique that uses magnetically labelled water molecules in the blood to trace cerebral blood flow (CBF). As CBF is directly linked to metabolic activity, ASL is a good non-invasive, radiationfree, low-cost marker of perfusion. Whole brain CBF maps can be calculated from the acquired data allowing group comparisons [74]. In PD, ASL has been shown to be an alternative to PET and SPECT for assessing perfusion [75,76]. ASL has not yet been applied to MSA, but recent experience in other neurodegenerative disorders, including various parkinsonian syndromes, is encouraging, suggesting that ASL may prove a useful and safe neuroimaging tool in the future.

Multimodal imaging and the future
Complex molecular processes such as neurodegeneration require comprehensive neuroimaging protocols to accurately describe and track disease progression and studies are now starting to combine multiple imaging modalities. Combining T2* relaxation rates with DTI metrics has revealed significant changes in the putamen of MSA compared with IPD. Comparing the clinical subtypes, the MSA-P group showed a higher MD in the putamen compared to IPD and MSA-C. Importantly, the combination of the two methods assessing structural integrity (T2* and MD) provided 96% accuracy in differentiating IPD cases from MSA-P [33]. In a group of early stage atypical parkinsonian syndrome patients with unknown diagnosis, a prospective study using both conventional T1/T2-MRI (regions of atrophy and signal changes) and DTI (MD and FA metrics) measures assessed the diagnostic accuracy when both techniques were used. A diagnosis was reached after clinical follow-up. As with the previous study, significantly higher MD in the putamen was present in the MSA-P compared to the rest of the group, and diagnostic accuracy increased when the DTI metrics were added [77]. Similarly, adding SWI data to conventional structural MRI improved the accuracy of identifying the MSA cases from the atypical parkinsonism group [48].
Although not yet applied to MSA, a combination of MRI, DTI, fMRI, and ASL has improved diagnostic accuracy in other neurodegenerative disorders, e.g., AD and FTD based on structural and functional white matter involvement [78]. A similar approach could provide significant qualitative and quantitative markers of disease and progression for MSA, where the white matter tracts are affected early in the disease course.

Conclusion
In recent years, there has been significant progress in neuroimaging techniques and their application to research into neurodegenerative disorders. In MSA, most studies in the past have focused on the volumetric and structural patterns of the disease. However, improved diagnostic accuracy and novel disease progression markers have been reported using new connectivity and functional techniques. None of these tools alone are able to provide all the necessary quantitative and qualitative measured outcomes. However, a multimodal approach using these innovative technologies as part of the diagnostic toolkit seems likely to offer the best path for future progress in both clinical diagnosis and research into MSA.


extfieldsofstudy: Biology Medicine

text:
Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

span, following a highly complex process of coordinated cell division and DNA segregation, and visualized as long ladders of Z-rings 22 . Eventually, chains of unigenomic spores are formed, which have a thick protective spore wall facilitating long-term survival in the environment. Though ftsZ null mutants are viable, they fail to produce septa and hence do not sporulate and cell division is not essential for growth of Streptomyces, which provides a unique system for the study of this process 23,24 .
Sporulation-specific cell division is controlled by the SsgA-like proteins (SALPs), which are exclusively found in morphologically complex actinobacteria 25,26 . The canonical view is that cell division is negatively controlled by the action of the Min system that inhibits division away from midcell, and by nucleoid occlusion (Noc) to avoid septum synthesis near the chromosome to avoid DNA damage, as seen in B. subtilis and E. coli. In contrast, cell division in streptomycetes is positively controlled by the recruitment of FtsZ to future septum sites by SsgB, in an SsgA-dependent manner 27 . As a consequence, both SsgA and SsgB are required for sporulation 28,29 . We recently showed that SepG (formerly called YlmG) assists in docking of SsgB to the membrane, and also plays a major role in maintaining the nucleoid shape in the spores 30 .
Many of the genes for the components of the divisome and the cell-wall biosynthetic machinery are located in the so-called dcw cluster (division and cell-wall synthesis 31 ). Most of these genes have been studied extensively and their functions have been well characterized. However, little is known of the genes ylmD and ylmE that lie immediately downstream of, and likely form an operon with, ftsZ on the genome of streptomycetes and many other bacteria, including firmicutes. Earlier work on a mutant of S. venezuelae lacking both ylmD and ylmE showed that the double mutant had no obvious phenotypic defects 32 .
In this study, we show that deletion of ylmE alone results in severe cell division defects, while deletion of ylmD only slightly affects sporulation. Interestingly, the cell division defects of ylmE mutants were rescued by the additional deletion of ylmD, and ylmDE mutants had no obvious sporulation defects. These data strongly suggest that expression of YlmD alone is detrimental for sporulation-specific cell division in streptomycetes, which is counteracted by YlmE. This is consistent with the phylogenetic evidence that some bacteria only harbor an ortholog of ylmE, whereas ylmD never occurs without ylmE.

Results and Discussion
Phylogenetic analysis of YlmE (SCO2080) and YlmD (SCO2081). Many of the genes in the dcw gene cluster have been extensively studied and their functions are well established. However, this is not the case for ylmD and ylmE, which lie immediately downstream of ftsZ in many bacteria. In all Streptomyces genomes analyzed, ftsZ (SCO2082), ylmD (SCO2081) and ylmE (SCO2080) form an operon, an observation that is supported by high-resolution transcript mapping 33 ; moreover there is apparent translational fusion between ftsZ and ylmD (overlapping start and stop codons) and only 6 nt spacing between ylmD and ylmE. This transcriptional coupling to ftsZ suggests that these genes may play a prominent role in the cell division process. Transcript levels of ftsZ are similar to those of ylmD and ylmE during vegetative growth on MM agar plates; however, transcription of ftsZ is enhanced during sporulation, while that of ylmD and ylmE is not significantly altered 34 . YlmD and YlmE are wide-spread in Gram-positive bacteria, and particularly in actinobacteria. SCO2081 and SCO2080 share 32% aa identity with YlmD and YlmE of B. subtilis, respectively. Orthologues of these proteins are also found in several genera of Gram-negative bacteria. Phylogenetic analysis of the YlmD and YlmE proteins in actinobacteria shows that while YlmE is widespread, YlmD is often absent in actinobacteria, such as Stackebrandtia, Catenulispora, Salinispora, Micromonospora, Amycolatopsis and Mycobacterium, several of which are spore-forming actinobacteria ( Fig. 1). Remarkably, there are no examples of YlmD being present in the absence of YlmE and thus it would appear that loss of YlmD (SCO2081) has occurred on multiple occasions, given that there is wide but patchy distribution of this gene across distinct actinobacteria lineages and the sequences clade tightly within the accepted actinomycete phylogenies.
Analysis of ylmD using the EMBL String engine 35 shows functional linkage in two groups to cell division associated genes (sepF (SCO2079), SCO2085 and ftsZ) along with a group of mainly hypothetical proteins (STRING Data link: http://bit.ly/2gc5kCB). YlmD has a domain that has homology to the multiple-copper polyphenol oxidoreductase laccases, which are oxidoreductases that are widely distributed in both prokaryotes and eukaryotes 36 . A similar analysis of YlmE also indicates a functional linkage to cell-wall biosynthesis and cell division (STRING Data link: http://bit.ly/2gbPyHK). YlmE is a member of the family of YBL036c-like proteins, which generally contain pyridoxal 5-phosphate dependent enzymes. The structure of YBL036c from Saccharomyces cerevisiae was resolved at 2.0 Å resolution (PDB 1CT5 37 ). The protein has homology to the N-terminal domains of alanine racemases but lacks of the β-sandwich domain which would likely limit the activity of YBL036c as alanine or non-specific amino acid racemase 38 . To test the hypothesis that YlmE may have alanine racemase activity and thus may play a role in determining the amino acid composition of the peptidoglycan, we tested purified YlmE for alanine racemase assays as described previously 39 , using alanine racemase Alr as positive control. Whereas purified Alr successfully catalyzed the conversion of L-alanine to D-alanine, YlmE could not perform this reaction under the same conditions, and over-expression of YlmE failed to restore a D-Ala prototrophy to alr null mutants (data not shown). These observations coupled with protein structure homology data make it highly unlikely that YlmE functions as an alanine racemase in vivo. ylmD and ylmE are required for proper sporulation. To analyze the role of ylmD and ylmE, deletion mutants were created in S. coelicolor M145 as detailed in the Materials and Methods section. The +25 to +696 region of ylmE (SCO2080) or the +25 to +705 region of ylmD (SCO2081) were replaced by the apramycin resistance cassette, followed by deletion of the cassette using the Cre recombinase so as to avoid polar effects. For each mutant, four independent mutants were selected and all had the same phenotype. Therefore, one was selected for more detailed analysis, designated GAL47 (S. coelicolor M145 ΔylmD) and GAL48 (S. coelicolor M145 ΔylmE). The M145 ylmDE mutant (GAL130) was created by replacing ylmD in ylmE null mutant GAL48 by the apramycin resistance cassette.
The ylmD null mutant GAL47 had a wild-type-like appearance, while ylmE null mutant GAL48 hardly produced any grey pigment after 5 days incubation on SFM agar plates, indicative of a failure to complete full development. Surprisingly, ylmDE mutant GAL130 had a grey appearance similar to that of the parental strain M145 ( Fig. 2A). Phase-contrast light microscopy of impression prints of the top of the colonies demonstrated that while the parent produced typical long spore chains, the ylmD null mutant produced abundant but aberrantly sized spores, the ylmE null mutant produced fewer spores and those produced were unusually large. The ylmDE double mutant produced abundant spores, though some were irregularly sized (Figs 2B and S1). The same defect in sporulation was observed on R5 and MM mannitol agar plates. Antibiotic production was not affected, and the mutants produced normal levels of the pigmented antibiotics actinorhodin and undecylprodigiosin.
To further ascertain that the sporulation defects were indeed solely due to the deletion of the respective genes, we introduced plasmids expressing ylmD or ylmD-egfp in the in the ylmD null mutant and ylmE or ylmE-egfp in the ylmE mutant, in all cases with transcription directed from the native ftsZ promoter region. While the majority of the spores of the complemented ylmD mutant had a regular appearance, those of the complemented ylmE mutants still showed variable lengths (Fig. 2B). This partial complementation suggests that the deletion of ylmE may have polar effect on the expression of its downstream genes. This is supported by the fact that introduction of a plasmid harboring the entire region from murD to divIVA fully restored sporulation to ylmE mutants (Figs 2B and S2). Promoter probing using the redD reporter system 40 showed that besides the intergenic promoter between ylmE and sepF, an additional promoter is located within ylmE, suggesting that deletion of the entire ylmE gene negatively affects the transcription of sepF (data not shown). This may explain why ylmE alone failed to fully complement the ylmE null mutant.
To quantify the spore lengths and their size distribution (i.e. variability), approximately 300 spores from the parental strain S. coelicolor M145, its ylmD and ylmE mutants and the genetically complemented strains were measured from light images from phase-contrast microscopy (100x magnification). Spore sizes were compared using a boxplot analysis (Fig. 2C). Wild-type spores typically had lengths between 0.8-1.2 µm, with an average length of 1.01 ± 0.15 µm, while ylmD mutant spores showed a slightly broader distribution (Fig. 2C), with an average length of 1.12 ± 0.27 µm. The average length was reduced to wild-type levels by complementation, namely to 1.03 µm and 1.05 µm by introduction of ylmD and ylmD-egfp, respectively. Spores of the ylmE null mutant The sporulation defect of the ylmD mutant could be complemented by introduction of wild-type ylmD and ylmD-eGFP, while complementation of the ylmE mutant by wild-type ylmE or ylmE-egfp restored sporulation, although irregularly sized spores were often produced. Full complementation of ylmE mutants was obtained by introduction of pKR8, which contains part of the S. coelicolor dcw cluster. Bar, 5 μm. (C) Size distributions of wild-type spores and those of ylmD, ylmE and ylmDE mutants presented as boxplots. Data are presented as median and interquartile range in boxplots, with whiskers spread to the maximal and minimal values. The numbers (n) of spores measured for each strain are indicated. Assessment of normality of spore length data was performed by a Kolgomorov-Smirnov test, the results showing that spore length data for all mutants followed non-normal distribution (Dmax > Dcrit). The shown two-tailed P values between each mutant and the parental strain were calculated using a Mann-Whitney U test. The two-tailed P values were far below 0.05, which shows that the sizes of the mutant spores deviated significantly from those of wild-type spores. were much larger and with a significantly wider variation, showing an average length of 2.23 ± 0.86 µm (Fig. 2C). Genetic complementation of the ylmE mutant restored spore length variation of ylmE mutant to wild-type levels, with an average size of 1.03 ± 0.19 µm. Partial complementation of the ylmE null mutant was seen when constructs expressing ylmE or ylmE-egfp were introduced into the mutant, with spore lengths of 1.62 ± 0.50 µm and 1.85 ± 0.59 µm, respectively. The average spore length of the combined ylmDE mutant was much closer to that of the parental strain, namely 1.23 ± 0.34 µm, thereby showing significantly smaller size variation than ylmE null mutants (Fig. 2C). The statistical validity of the variations in spore sizes between mutants and the parental strain was validated by a Kolgomorov-Smirnov test and a Mann-Whitney U test, which confirmed that the sizes of mutant spores deviated significantly from those of wild-type spores.
To study the spores of ylmD and ylmE null mutants at high resolution, cryo-scanning electron microscopy (SEM) was performed, which again demonstrated the sporulation defects. The parental strain S. coelicolor M145 produced abundant spore chains, with nearly all hyphae fully developed into mature spore chains (Fig. 3A,B). The ylmD null mutant GAL47 frequently produced aberrantly sized spores (Fig. 3C,D), while in ylmE null mutant GAL48, precious few and often aberrant spores were identified (Fig. 3E,F). The same approach was used to create ylmD and ylmE mutants in Streptomyces lividans 66, and these again showed the same morphology, strongly supporting the notion that the observed phenotypes were solely due to the mutations in ylmD or ylmE (data not shown). The highly similar phenotypes of the S. coelicolor and S. lividans mutants support the notion that the genes play a key role in sporulation-specific cell division and are consistent with the conserved function of the genes in sporulation-specific cell division in Streptomyces.
Deletion of ylmD and especially ylmE results in reduced robustness of spores. Viability of the spores was tested by plating around 1000 spores -counted in a hemocytometer -of S. coelicolor M145 and its mutants GAL47 (ΔylmD) and GAL48 (ΔylmE) onto SFM agar plates. While the wild-type strain had close to 100% viability, spores of the ylmD and ylmE mutants had reduced viability, with 50% and 60% viable spores, respectively. Spores of in particular the ylmE mutant were heat sensitive, with only 11 ± 1% viability after 15 min incubation at 58 °C, as compared to 47 ± 6% survival for ylmD null mutant spores and 55 ± 5% for the parental strain. Genetic complementation of the ylmE null mutant restored wild-type survival to heat shock (58 ± 3%). The same was true when ylmD was deleted in the ylmE null mutant (56 ± 4% survival). To analyze the possible cell-wall defects in more detail, all strains were grown on SFM agar plates for 5 days and analyzed by Transmission Electron Microscopy (TEM) (Fig. 4). The parent produced typical spore chains and thick-walled spores. Conversely, ylmD and ylmE mutant spores were deformed and highly irregular. In particular the spores of the ylmE mutant had a thin wall similar to that of the hyphae, suggesting that spore-wall synthesis was compromised.
YlmD and YlmE are required for peptidoglycan synthesis at the septum. To analyze cell wall and membrane distribution in ylmD and ylmE mutants, fluorescence microscopy was performed on five-days old SFM surface-grown cultures of mutants GAL47 (M145 ΔylmD) and GAL48 (M145 ΔylmE) and compared to the parental strain M145. Peptidoglycan precursors were stained with FITC-WGA or Oregon-WGA and membranes visualized by staining with FM5-95. In wild-type pre-division aerial hyphae, long symmetrical septal ladders were observed when stained for cell wall or membranes (Fig. 5A). In contrast, aerial hyphae of the mutants showed highly disturbed cell wall and membrane distribution, which was more pronounced in ylmE than in ylmD mutants (Fig. 5A). Septation of ylmD mutants was complemented via re-introduction of ylmD; similarly, septation was restored to the ylmE mutant by re-introduction of ylmE, although peptidoglycan synthesis and septum spacing was still irregular (Fig. 5B). These data again show that the consequences of deleting ylmE are much more severe for sporulation-specific cell division then when ylmD is deleted.

Cellular localization of YlmD and YlmE. Given the impact of ylmDE on sporulation, we analyzed how
YlmD and YlmE were localized in the hyphae of S. coelicolor. To this end, constructs based on the low-copy number vector pHJL401 were prepared to allow the expression of YlmD-eGFP and YlmE-eGFP fusion proteins, which were expressed from the natural ftsZ promoter region (see Materials and Methods section for details). These constructs were then introduced into S. coelicolor M145 and (as a control for functionality) in the mutants. The constructs partially restored development to the respective mutants, with significant restoration of the sporulation defects in ylmD mutants, and partial restoration of sporulation to ylmE mutants (Fig. 2B).
In vegetative hyphae, only very weak signals were obtained for YlmD-eGFP and YlmE-eGFP, indicative of low protein expression (Fig. 6A). In early and late aerial hyphae, both YlmD-eGFP and YlmE-eGFP became more visible and showed diffuse localization along the wall of the aerial hyphae (Fig. 6A). During early sporulation, YlmD-eGFP and YlmE-eGFP showed an irregular pattern of varying intensity, with some spores showing bright fluorescence indicative of high concentrations of YlmD or YlmE, while others hardly showed any fluorescence (Fig. 6A). This suggests that YlmD and YlmE are differentially expressed throughout the nascent spore chains, and do not exclusively co-localize with FtsZ and the divisome. To rule out possible proteolysis of YlmD-or YlmE-eGFP fusion proteins, Western analysis was performed on extracts of surface-grown S. coelicolor mycelia, using antibodies against GFP. S. coelicolor M145 with empty vector and S coelicolor M145 expressing freely mobile eGFP 41 were included as controls. Only a single band was identified in strains GAL50 and GAL49, corresponding to the expected lengths for YlmD-eGFP and YlmE-eGFP, respectively (Fig. S3). In both cases, the predicted mass of the fusion protein is around 52 kDa, which conforms well to the apparent mobility seen by Western analysis. The strain expressing only eGFP showed the expected band of around 27 kDa for the non-fused protein. Therefore, we conclude that the observed localizations can indeed be ascribed to intact YlmD-or YlmE-eGFP.
The fluorescence intensity of YlmD-eGFP and YlmE-eGFP was measured on images representing different developmental stages (Fig. 6B). As development progressed, the fluorescence intensities increased for both YlmD-eGFP and YlmE-eGFP and reached peak levels during the phase corresponding to late aerial growth and early sporulation. When spores matured, YlmD-eGFP and YlmE-eGFP signals decreased to the level as in vegetative growth. These results suggest YlmD and YlmE proteins are active primarily during the phase of sporulation-specific cell division.

Localization of FtsZ is disturbed in ylmD and ylmE null mutants. To investigate the localization of
FtsZ in the mutants, integrative plasmid pKF41 that expresses FtsZ-eGFP from the native ftsZ promoter region 42 was introduced into the ylmD and ylmE mutants, to generate strain GAL52 and GAL53, respectively. Prior to sporulation, typical ladders of Z-rings were observed in the parental strain S. coelicolor M145, while ylmD and ylmE null mutants showed abnormal Z-ladders (Fig. 7). In the absence of YlmD, ladders were still observed, but the intensity varied and spacing between the individual rings was less regular, with many neighboring Z-rings either close together or widely spaced. Consistent with the sporulation defect, ylmE null mutants produced very Wild-type spores (M145) show regular sizes and appearance (A). In contrast, spores of the ylmD (B) and ylmE (C) null mutants have an irregular appearance. Note the lighter appearance of the spore wall in the ylmD null mutant and the lack of the typical thick spore wall in ylmE mutants. Cultures were grown on SFM agar plates for 5 days at 30 °C. Bar, 500 nm.
few Z-rings, and the few Z-ladders that where formed were highly irregular or unfinished, and significantly shorter than in the parental strain. Thus, FtsZ localization is irregular in ylmD mutants and highly compromised in ylmE mutants.  variable lengths. Surprisingly, the additional deletion of ylmD greatly rescued the sporulation-deficient phenotype of ylmE mutants, with the ylmDE null mutant producing abundant and mostly regularly sized spores.
We propose that YlmD and YlmE form a two-part system, reminiscent of toxin-antitoxin systems 43 . Similarly dysfunction of one protein in the presence of another has also been reported for the sporulation control protein WhiJ, following the observation that media-dependent sporulation defects caused by a point mutation in whiJ (SCO4543) could be rescued by the full deletion of whiJ 44 . The authors proposed a model wherein repression of developmental genes by WhiJ could be released when WhiJ interacts with a WhiJ-associated protein encoded by the adjacent gene SCO4542 44 . It was suggested that the aberrant protein WhiJ* still binds to operator sequences of developmental genes, leading to their transcriptional repression, but fails to associate with SCO4542, with a permanent block of transcription of developmental genes as a result 44 . Similarly, the expression of YlmD alone is detrimental for sporulation, suggesting that YlmD acts in a deleterious manner towards sporulation, yet this effect can be relieved by the presence of YlmE. Supporting evidence for this is that deletion of both genes allows Streptomyces to develop normally, and by the observation that ylmD is never found alone in bacterial genomes, while the occurrence of an orphan ylmE is seen frequently. Further support comes from the report that ylmDE double mutants of S. venezuelae also sporulate relatively normally 32 . This also indicates that this two-part system functions in a similar manner in a range of Streptomyces species. This apparently rules out major divergence between the two morphologically distinct Streptomyces clades, namely those that sporulate in submerged cultures and those that do not 26,45 . However, it will be very interesting to see if submerged sporulation of S. venezuelae is prevented by the deletion of just ylmE. Currently, we are performing detailed structural and functional analysis of YlmD and YlmE, to elucidate their biochemical function and their precise role in bacterial cell division.

Methods
Phylogenetic analysis of ylmD and ylmE. The amino acid sequence of YlmD and YlmE were extracted from StrepDB (http://strepdb.streptomyces.org.uk) and used to search the NCBI database (www.ncbi.nlm.nih. gov) using BLASTP against the non-redundant protein sequence database. Alignment of YlmD and YlmE was generated using ClustalW 46 followed by manual editing in MEGA v. 4.0. The neighbour-joining trees 47 were generated with default parameters settings as implemented in MEGA v. 4.0 48 . The maximum-likelihood trees were made using the best fit models predicted by MEGA. Tree reliability was estimated by bootstrapping with 1000 replicates. Trees were drawn with either N-J or ML algorithms gave trees of similar topologies indicating that the phylogenies are likely to reliable.
Bacterial strains and media. All bacterial strains used in this study are listed in Table S1. E. coli JM109 was used for routine cloning and ET12567 49 to prepare nonmethylated DNA to bypass the methyl-specific restriction system of S. coelicolor. E. coli strains were propagated in Luria broth, where appropriate supplemented with antibiotics for selection, namely ampicillin (100 µg/ml end concentration), apramycin (50 µg/ml) and/or chloramphenicol (25 µg/ml). S. coelicolor A3(2) M145 50 and S. lividans 66 51 were obtained from the John Innes Centre strain collection. S. coelicolor strains were grown on soya flour medium (SFM) or minimal media mannitol (MM) agar plates for phenotypic characterization and on R5 agar plates for regeneration of protoplasts 52 . Antibiotics used for screening Streptomyces were apramycin (20 µg/ml end concentration) and thiostrepton (10 µg/ml).
Plasmids and constructs. All plasmids and constructs described in this study are summarized in Table S2.
The oligonucleotides used for PCR are listed in Table S3. PCR reactions were performed using Pfu DNA polymerase as described 53 . All inserts of the constructs were verified by DNA sequencing, which was performed at BaseClear (Leiden, The Netherlands).
Constructs for the deletion of ylmD and ylmE. The strategy for creating knock-out mutants is based on the unstable multi-copy vector pWHM3 54 as described previously 55 . For each knock-out construct roughly 1.5 kb of upstream and downstream region of the respective genes were amplified by PCR from cosmid St4A10 that contains the dcw cluster of S. coelicolor. The upstream region was thereby cloned as an EcoRI-XbaI fragment, and the downstream part as an XbaI-BamHI fragment, and these were ligated into EcoRI-BamHI-digested pWHM3 (for the precise location of the oligonucleotides see Table S3). In this way, an XbaI site was engineered in-between the flanking regions of the gene of interest. This was then used to insert the apramycin resistance cassette aac(3) IV flanked by loxP sites, using engineered XbaI sites. The presence of the loxP recognition sites allows the efficient removal of the apramycin resistance cassette following the introduction of a plasmid pUWL-Cre expressing the Cre recombinase 56,57 . Knock-out plasmids pGWS728 and pGWS729 were created for the deletion of nucleotide positions +25/+ 696 of ylmE (SCO2080) and +25/+ 705 of ylmD (SCO2081), whereby + 1 refers to the translational start site of the respective genes. This allowed first the replacement by the apramycin resistance cassette. Subsequently the apramycin resistance cassette was removed using expression of pUWLCre. To create a ylmDE double deletion mutant, construct pGWS1044 was created which contains the upstream region of ylmD and downstream region of ylmE flanked by loxP sites, and with the apramycin resistance cassette aac(3)IV inserted in between.
For complementation of the ylmE and ylmD null mutants, the entire coding regions of SCO2080 and SCO2081 (with stop codons) were amplified from the S. coelicolor M145 chromosome using primer pairs ylmE_F + 1 and ylmE_R + 723 and ylmD_F + 1 and ylmD_R + 732, respectively. The PCR products were digested with StuI/ XbaI, and inserted downstream of the native ftsZ promoter region in pHJL401, respectively. Thus constructs pGWS1042 and pGWS1043 were generated that express ylmE and ylmD, respectively, under control of the S. coelicolor ftsZ promoter region. Alternatively, construct pKR8 was used for complementation; pKR8 is based on integrative vector pIJ8600 58 and contains the 2227742-2241015 region of the S. coelicolor genome, encompassing the end of murX (SCO2087), the entire coding sequences of murD, ftsW, murG, ftsQ, ftsZ, ylmD, ylmE, sepF, sepG, divIVA and a large part of SCO2076 (encoding Ile-tRNA synthetase).
Constructs for the localization of YlmD and YlmE. The entire coding regions of SCO2080 and SCO2081 (without stop codons) were amplified from the S. coelicolor M145 chromosome using primer pairs ylmE_F + 1 and ylmE_R + 717 and ylmD_F + 1 and ylmD_R + 726, respectively. The PCR products were digested with StuI/ BamHI, and inserted downstream of the native ftsZ promoter region and immediately upstream of egfp in pHJL401. The latter is a highly stable vector with low copy number that generally results in wild-type transcription levels and is well suited for among others complementation experiments 40 . Thus constructs pGWS757 and pGWS758 were generated that express YlmE-eGFP and YlmD-eGFP, respectively, from the native S. coelicolor ftsZ promoter region. Plasmid pKF41 expresses FtsZ-eGFP from its own promoter region 42 . Constructs pGWS757 and pGWS758 were also used to complement ylmE and ylmD null mutants, respectively. eGFP detection using western blot analysis. Strains were grown on SFM agar plates containing the appropriate antibiotics overlaid with cellophane disks. Biomass (mycelium and spores) was collected after 6 days Scientific RepoRts | (2018) 8:7328 | DOI:10.1038/s41598-018-25782-1 of growth at 30 °C and suspended in urea lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF and 65 mM DTT), followed by cell disruption via sonication. Western blots were performed as described 59 . Briefly, cell lysates were obtained after centrifugation at 30,000 × g for 30 min at 4 °C. Proteins were separated on a 12.5% SDS-PAGE gel, and then transferred to PVDF membrane using Mini Trans-Blot (Bio-Rad). As primary antibody a 1:2000 dilution of GFP antibody (ThermoFisher, REF A11122) was used and incubated for 16 h at 4 °C in the presence of 3% BSA. After washing, the membrane was incubated with alkaline phosphatase conjugated anti-rabbit IgG (Sigma) as secondary antibody for 1 h at room temperature. Visualization was done using BCIP/ NBT color development substrate.
Microscopy. Fluorescence and light microscopy were performed as described previously 60 . For peptidoglycan staining we used FITC-labeled wheat germ agglutinin (FITC-WGA) or Oregon Green 488 conjugated WGA (Oregon-WGA); for membrane staining, we used FM5-95 (all obtained from Molecular Probes). All images were background-corrected, setting the signal outside the hyphae to 0 to obtain a sufficiently dark background. These corrections were made using Adobe Photoshop CS4. Cryo-scanning electron microscopy (cryo-SEM) and transmission electron microscopy (TEM) were performed as described 61 . For quantification of fluorescence intensity of YlmD-eGFP and YlmE-eGFP, the arbitrary average intensity values of manually selected areas of interest were determined as relative fluorescence intensity. All green fluorescent images were made with equal exposure time. The difference (D n ) between the observed cumulative distribution function (F obs ) and the expected cumulative distribution function (F exp ) was calculated as: D n = |F exp (x) − F obs (x)|. The maximal value was identified as D max . The null hypothesis that the data comes from the specified distribution was rejected if D max > D crit . The Mann-Whitney U test was done using the SPSS software release 24.0.
Computer analysis. DNA and protein database searches were completed by using StrepDB (http://strepdb. streptomyces.org.uk/). Phylogenetic analysis was done using the STRING engine at EMBL (www.string.EMBL).
Data availability. All strains, materials and data will be made available upon request.


s2fieldsofstudy: Biology Medicine

text:
Downregulation of microRNA-17-5p improves cardiac function after myocardial infarction via attenuation of apoptosis in endothelial cells

MicroRNA-17-5p (miR-17-5p) was indicated to suppress the formation of blood vessels, which is associated with cardiac function after myocardial infarction. In this study, the relationship between miR-17-5p and cardiac function was researched. Human umbilical vein endothelial cells were infected with adenoviruses. Apoptosis was determined by Annexin V-7AAD/PI. Real-time RT-PCR was used to evaluate miR-17-5p and ERK levels. Western blotting was used to determine the levels of ERK, the anti-apoptosis protein bcl-2 and apoptosis proteins, including bax, caspase 3, and caspase 9. An in vivo acute myocardial infarction (AMI) model was established in SD male rats. Heart function was evaluated by echocardiography prior to inducing AMI and after 7 and 28 days later. The heart was removed to perform histological examination, real-time RT-PCR, and western blotting, as described above. The result indicated that the ERK pathway was activated by miR-17-5p downregulation and an increase in the level of the anti-apoptosis protein bcl-2; however, the levels of apoptosis proteins (bax/caspase 3/caspase 9) were decreased. The results were completely reversed when miR-17-5p was up-regulated. At 7 and 28 days after the induction of AMI, in the miR-17-5p inhibition group, the infarction areas and collagen fibers were decreased, apoptosis in cardiac tissues was inhibited, and the endothelial growth process was promoted. Therefore, MiR-17-5p silencing protects heart function after AMI through decreasing the rate of apoptosis and repairing vascular injury.

Introduction
Myocardial infarction is one of the leading causes of death globally and is mainly characterized by vascular endothelial injury due to apoptosis, cardiac fibrosis, inflammation, and pathological remodeling (Du et al. 2014). Though precautions and medical treatments for myocardial infarction have improved, a considerable number of deaths due to myocardial infarction occur every year . Heart failure after myocardial infarction (MI) is closely associated with microcirculation (Al Suwaidi et al. 2000;Joner et al. 2006;Farb and Boam 2007;Dangas et al. 2010). Studies have confirmed that the common pathological physiological basis of myocardial infarction is delayed repair of the damaged vascular endothelium (Finn et al. 2007;Inoue et al. 2011). It is highly important to completely restore endothelial function soon after myocardial infarction.
MicroRNA is a newly discovered class of small nonprotein-coding mRNAs, which have rapidly became known as critical regulators of gene expression (Krol et al. 2010). Mature microRNAs negatively regulate gene expression through two mechanisms: they prevent protein synthesis at the translational level and cause degradation of the mRNA of the target gene (van Rooij 2011). Every microRNA can control the expression of multiple genes simultaneously, which makes the translation of traditional single locus Communicated by S. Hohmann.
* Hong Jiang phy_hongj@163.com 1 3 control to multiple site intervention possible (van Rooij 2011). Cell-specific expression is another major characteristic of microRNAs (van Rooij 2011). Through post-transcriptional regulation, microRNAs define the proteomes of cells by regulating gene expression, and their regulatory activities are involved in various cellular functions and generate specific biological phenotypes (Norbury 2010). Recently, many miRNAs have been confirmed to be involved in the regulation of the pathological and physiological processes of heart disease (Ren et al. 2009;Wang et al. 2010;Pan et al. 2012).
Researchers have shown that microRNA-17 (miR-17) is highly expressed in endothelial cells (EC) (Bonauer et al. 2009) and has low expression in vascular smooth muscle cells (VSMC). Researchers have also found that miR-17 exerts an anti-angiogenic function; therefore, inhibiting its expression can improve the density of EC angiogenesis in vitro (Doebele et al. 2010;Yu et al. 2010). Other studies have indicated that in the process of rat carotid artery injury and repair, the expression of miR-17 rises continuously, while the expression of miR-18a and miR-19a, which are known to promote angiogenesis, show no obvious changes (Ji et al. 2007). Here, we hypothesized that downregulation of miR-17 would enhance cardiac function after MI by improving endothelial function and increasing the density of new blood vessels.

Cell culture and adenovirus infection
Human umbilical vein endothelial cells (HUVECs) were purchased from Sciencell Company (Cat. No. 8000) and cultured with endothelial cell medium (ECM) (Sciencell Cat. No. 1001) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement (ECGS), and 1% penicillin/streptomycin solution (P/S). All HUVECs were used for experiments between the first and fifth passages. Recombinant adenoviruses containing pre-miR-17 or Antagomir-17 for miR-17-5p overexpression (Ad-Pre-miR-17) and miR-17-5p silencing (Ad-Antagomir-17), respectively, were prepared. Ad-NC-Pre-miR-17, which contains the same gene sequence as Pre-miR-17, but does not affect the expression of miR-17-5p, was used as the control adenovirus for Ad-Pre-miR-17. Ad-NC-Antagomir-17, which contains the same gene sequence as Antagomir-17, but does not affect the expression of miR-17-5p, was used as the control adenovirus for Ad-Antagomir-17. For adenovirus infection, the above-mentioned HUVECs were incubated with Ad-Pre-miR-17/Ad-NC-Pre-miR-17 or Ad-Antagomir-17/Ad-NC-Antagomir-17 at a multiplicity of infection (MOI) of 30 in serum-free media for approximately 4 h. Then, the media were removed, and the cells were incubated with complete medium for the next 72 h.

Apoptosis analysis
Cell apoptosis was measured in an Annexin V-7AAD/propidium iodide (PI) assay. HUVECs infected with the recombinant adenoviruses mentioned above were divided into three parts and seeded in 60-mm plates (1 × 10 5 ). After synchronization in ECM with 0.5% FBS for 24 h, one part was cultivated in ECM containing 5% FBS for 24 h, and the second part underwent apoptosis in ECM without FBS for 24 h, and the third part treated with ERK inhibitor PD098059 at a concentration of 10 µM in ECM without FBS for 24 h. Cells were collected and re-suspended in 1× binding buffer. Apoptotic cells were measured with an Annexin V-7AAD apoptosis detection kit (BD Biosciences, USA) according to the manufacturer's protocol. Five microliters of Annexin V-7AAD and 5 µl of PI were added to the re-suspended cells, and the cells were incubated for 15 min at room temperature in the dark. Apoptosis was measured using a BD accuriC6 flow cytometer, and the data were processed using FlowJo (FlowJo, Ashland, OR, USA) software.

Western blotting
For whole-cell protein extraction, HUVECs under apoptosis induction were washed three times with cold PBS and subsequently lysed in cold RIPA lysis buffer (20 mM Tris Cl, pH 7.4/137 mM, NaCl/2 mM EDTA/1% Triton/10% glycerol/25 mM β-glycerol phosphate/1 mM PMSF, and protease inhibitor mixture). Cell or heart lysis was performed on ice for 30 min, and clear protein extracts were obtained by centrifugation at 12,000×g for 10 min. Protein concentration was determined with the bicinchoninic acid protein assay (Bipec). Proteins were separated by SDS-polyacrylamide gel electrophoresis and were transferred to PVDF membranes. For immunoblotting, PVDF membranes were blocked with 5% milk and probed with antibodies against total ERK and phospho-ERK, bax, bcl-2, caspase 3, caspase 9 or GAPDH (all from Abcam) overnight at 4 °C. After washing the membranes with TBST three times (10 min each time), they were incubated with peroxidase-conjugated secondary antibodies (BOSTER) for 2 h at room temperature. The proteins were detected using the ECL Plus detection kit (Bipec).

SD rat AMI model
All animals used in this study were provided and cared for by the Animal Center of Renmin Hospital of Wuhan University. The experimental procedures and animal care were approved by the Animal Care and Use Committee of Wuhan University. All animals were given a conventional diet until they were sacrificed. All of the animal protocols complied strictly with the Institutional Animal Care and Use Committee guidelines. The procedure for inducing myocardial infarction (MI) in rats was described previously. Briefly, male Sprague-Dawley (SD) rats (200-250 g) were anesthetized with pentobarbital (30 mg/kg intraperitoneal injection). The adenoviruses (5 × 10 9 /100 μL) mentioned above were injected into the apex of the heart after the left anterior descending branch (LAD) was ligatured. The rats were euthanized at 7 or 28 days after MI, and the tissues were harvested for specific protocols. The rats were divided into four groups with five rats in each group: group 1: Ad-NC-Antagomir-17 for 7 days, group 2: Ad-Antagomir-17 for 7 days, group 3: Ad-NC-Antagomir-17 for 28 days, and group 4: Ad-Antagomir-17 for 28 days. The sham groups were exposed to the surgery without AMI and continued to be fed for 7 or 28 days. Cardiac function was evaluated by echocardiography before surgery and was re-evaluated at 7 or 28 days after MI. For histology, the tissues were perfusion-fixed with buffered formalin phosphate, and the hearts were harvested and processed. For real-time RT-PCR and western blot, the tissues were freshly removed and snapfrozen, as described above.

Morphometric analysis
At 7 or 28 days after the operation, the hearts were harvested, fixed in 4% paraformaldehyde, and then embedded in paraffin. For morphologic analysis, cardiac tissue at the infarction border area was removed. The infarction areas were determined by HE staining. Masson staining was used to examine collagen fiber areas. Apoptosis of cardiac tissue was determined by TUNEL staining; data are presented as percentages of total cells that are positive for apoptosis within a given area. CD31 staining was used to evaluate microcirculation formation; the perimeters of the areas positive for CD31 were measured, and the percentages of the total perimeters were also calculated.

Statistical analysis
Data are presented as means ± SEM. All values were analyzed using Student's t test for comparisons between two groups. A p value of < 0.05 was considered statistically significant.

Inhibition of miR-17-5p attenuates HUVEC apoptosis induced by culturing in ECM without FBS
After infection with adenoviruses, the level of miR-17-5p in the HUVECs was determined by real-time RT-PCR to evaluate the transfection efficiency. The miR-17-5p level was downregulated by 23% in the Ad-Antagomir-17 group and was up-regulated by 2.7-fold in the Ad-Pre-miR-17 group. Mir-17-5p silencing inhibited apoptosis in apoptosisinduced HUVECs. Inversely, up-regulation of miR-17-5p promoted apoptosis in apoptosis-induced HUVECs. However, in HUVECs cultured with ECM containing 5% FBS, the apoptosis ratios were low in both the miR-17-5p overexpression and miR-17-5p silencing HUVECs. To further assess whether apoptosis inhibition by miR-17-5p depends on the expression of ERK in HUVECs, the inhibitory effect of the ERK inhibitor PD098059 was evaluated. Treatment with PD98059 did not inhibit HUVEC apoptosis after miR-17-5p silencing in vitro (Fig. 1).

Mir-17-5p silencing stimulates ERK-signaling pathway
To further explore the mechanism of miR-17-5p on apoptosis, several key apoptosis pathways were examined. In apoptosis-induced HUVECs, the ERK pathway was stimulated by miR-17-5p silencing, as indicated by increased mRNA level of ERK and protein levels of ERK and phosphorylated ERK (P-ERK). ERK activity was evaluated by calculating P-ERK/ERK (Fig. 2).

Mir-17-5p silencing inhibits apoptosis through the ERK signaling pathway
To further investigate the effect of miR-17-5p on apoptosis via ERK pathway, several key downstream proteins were examined. In apoptosis-induced HUVECs, the protein level of the anti-apoptosis protein bcl-2 increased, and the levels of apoptosis proteins, including bax, caspase 9, and caspase 3, were reduced by miR-17-5p silencing; the opposite effect was observed with miR-17-5p overexpression (Fig. 3).

Inhibition of miR-17-5p inhibits apoptosis of rat heart after AMI via the ERK pathway
As shown in Figs. 4 and 5, miR-17-5p levels were decreased by 19.7 and 41.2%, respectively, in hearts from rats in the Ad-Antagomir-17 group and the Ad-NC-Antagomir-17 group at 7 or 28 days after the establishment of MI. Subsequent analysis of the protein expression levels of the apoptosis proteins mentioned above demonstrated a blunted response, and the anti-apoptosis protein bcl-2 demonstrated an intense response following miR-17-5p silencing compared to the control group at 7 or 28 days. This anti-apoptosis effect of miR-17-5p silencing was accompanied by increased mRNA expression levels of ERK and P-ERK, as observed by western blot.

Effect of miR-17-5p on histology of rat heart after MI
After 7-or 28-day MI, as shown in Fig. 6, HE staining showed that infarction areas were decreased by 43.7 and 73.2% in the Ad-Antagomir-17 group and Ad-NC-Antagomir-17 group, respectively (p < 0.05). Masson staining also indicated that areas of collagen fibers were reduced by 53.3 and 61.1% after miR-17-5p silencing in the Ad-Antagomir-17 group and Ad-NC-Antagomir-17 group, respectively (p < 0.05). Therefore, downregulation of miR-17-5p could protect cardiac structure of rat heart after AMI.

Effect of miR-17-5p on immunohistochemistry of rat heart after MI
After 7-or 28-day MI, as shown in Fig. 7, apoptosis of cardiac tissue cells was examined by TUNEL staining, which Fig. 1 MicroRNA-17-5p inhibition suppressed HUVEC apoptosis. a HUVECs were infected with adenovirus for 4 h and cultured with ECM containing 5% FBS for 72 h. Total mRNA was extracted, and the miR-17-5p level was detected by real-time RT-PCR (n = 3). b HUVECs infected with adenoviruses were divided into two parts; one part underwent apoptosis induction via incubation with ECM lacking FBS for 24 h, and the other part was cultured with ECM containing 5% FBS. Quantification of cellular apoptosis was performed with an Annexin V-7AAD/PI assay. c Apoptosis of HUVECs pretreated with the ERK inhibitor PD98059 (10 µM) was evaluated by an Annexin V-7AAD/PI assay (*p < 0.05 vs. Ad-NC-Pre-miR-17, # p < 0.05 vs. Ad-NC-Antagomir-17, n = 3) ▸ showed that downregulation of miR-17-5p inhibited the apoptosis of cardiac tissue cells compared to the Ad-NC-Antagomir-17 group (0.23 ± 0.101 vs. 0.61 ± 0.056 at 7 days after MI, 0.20 ± 0.0108 vs. 0.41 ± 0.038 at 28 days after I, p < 0.05). CD31, a marker of microcirculation formation, was stained and also indicated that miR-17-5p silencing promotes endothelial growth after MI compared to the Ad-NC-Antagomir-17 group (0.814 ± 0.047 vs. 0.360 ± 0.166 at 7 days after MI, 0.736 ± 0.069 vs. 0.432 ± 0.181 at 28 days after MI, p < 0.05). Thus, miR-17 silence could promote microcirculation of rat heart after AMI.

Effect of miR-17-5p on rat heart function after MI
At 7 or 28 days after MI, cardiac function was evaluated morphologically and quantitatively. Many index were used to determine the cardiac function, like ejection fractions (EF), fractional shortening (FS), end diastolic volume (EDV), End systolic volume (ESV), left ventricular internal dimension diastole (LVIDD), left ventricular internal dimension systole (LVIDS). As shown in Table 1, echocardiography revealed that downregulation of miR-17-5p enhanced heart function. The data mean percent decrease compared with heart function before AMI. Heart function was evaluated by echocardiography before the models were built and at 7 and 28 days later. The percentages of heart function decrease in each group were calculated (*p < 0.05 vs. Ad-NC-Antagomir-17).

Discussion
Many miRNAs have been reported to regulate senescence positively or negatively, but most studies have focused on the effect of miRNAs on the senescence of tumor cells at cellular level, or investigated the role of miRNAs in lower organisms (Du et al. 2015). Uniquely, our study systematically describes the functions of one specific miRNA, miR-17. MicroRNA-17 has been investigated in many fields, such as B-cell lymphoma (Lu et al. 2011), acute organ-specific autoimmune disease (Battistella et al. 2015), glucocorticoid-induced osteoclast differentiation and function (de Kouchkovsky et al. 2013), and so on. It is involved in angiopoiesis (Shi et al. 2014), apoptosis (Shan et al. 2013), and Fig. 2 Effects of miR-17-5p on the ERK pathway. a HUVECs were infected with adenovirus for 4 h and cultivated with ECM containing 5% FBS for 72 h. Then, they were cultivated with ECM without FBS for another 24 h to induce apoptosis. The cells were collected, and the protein levels of ERK and P-ERK were measured. b Protein levels of ERK, P-ERK in the HUVECs mentioned above were examined by western blot, and ERK activity was evaluated by calculating P-ERK/ ERK. c mRNA levels of ERK were examined by PCR (*p < 0.05 vs. Ad-NC-Pre-miR-17, # p < 0.05 vs. Ad-NC-Antagomir-17, n = 3) ▸ 1 3 Fig. 3 Effect of miR-17-5p on apoptosis-related protein levels. a HUVECs were infected with adenovirus for 4 h and cultivated with ECM containing 5% FBS for 72 h. Then, they were cultivated with ECM without FBS for another 24 h to induce apoptosis. The cells were collected, and the protein levels of apoptosis-related protein were measured. b Protein levels of anti-apoptosis protein bcl-2 were measured. c Levels of apoptosis protein Bax was measured. d Relative protein levels of apoptosis protein cas-pas3. e Relative protein levels of apoptosis protein caspas9 (*p < 0.05 vs. Ad-NC-Pre-miR-17, # p < 0.05 vs. Ad-NC-Antagomir-17, n = 3) proliferation (Honegger et al. 2015) in tumor tissues. However, the relationship between miR-17 and cardiovascular disease and the mechanisms underlying that relationship are still unknown. It is reported that miR-17 is sufficient to induce cardiomyocyte proliferation, but have nothing to do with cardiomyocyte apoptosis (Chen et al. 2013). However, this study focused on the effect of miR-17 on the apoptosis of ECs induced by serum deprivation. Our results indicated that miR-17-5p silencing had a greater apoptosis inhibition effect on ECs under serum-deprived conditions and had a nearly insignificant effect on ECs under normal conditions. The apoptosis rate is low under normal circumstances; thus, the apoptosis mechanism could not be triggered in the cells grown under normal conditions. These results show that miR-17-5p plays an important role in making life or death choices in ECs when apoptosis is triggered.
One major question being investigated in this study was how miR-17-5p silencing decreases the apoptotic rate of ECs in response to serum deprivation. Apoptosis can be triggered by extracellular stimuli via the death receptor and mitochondrial-mediated pathways (Scarabelli et al. 2002). The ERK pathway plays an important role in several cellular processes, such as cell survival, cell proliferation and apoptosis. It has been reported that activated ERK is responsible for high levels of cell growth in various cancers (Dhillon et al. 2007;Yang et al. 2013). Meanwhile, inhibition of activated ERK is associated with apoptosis (Salminen et al. 2010). Inhibition of miR-17-5p promotes the expression of activated ERK both in vivo and in vitro. Thus, we hypothesized that downregulation of miR-17-5p would decrease the apoptotic rate of endothelial cells both in vivo and in vitro. Proteins of the bcl-2 family play a pivotal role in apoptosis (Baell and Huang 2002;Goodsell 2002). BCL-2 interacting mediator of cell death (BIM) can be phosphorylated by members of mitogen activated protein kinase family. Extracellular signalregulated kinase 1/2 (ERK1/2)-mediated phosphorylation stimulates BIM degradation via the proteasome (Luciano et al. 2003;Kennedy et al. 2017). Bcl-2 is thought to be an anti-apoptotic protein and can inhibit the activity of the caspase cascade after it is initiated by the mitochondrial release of cytochrome c (Rosse et al. 1998;Cao et al. 2015). In contrast, bax is thought to be a proapoptotic protein of the bcl-2 family (Sedlak et al. 1995;Kluck et al. 1997), which can result in permeabilization of mitochondria (Rosse et al. 1998;Cao et al. 2015). We also detected the protein levels of caspase 3 and caspase 9, which, when in their active state, play a crucial role in the final step of apoptosis (Shebaby et al. 2014). Our research found that downregulation of miR-17 increased the protein level of bcl-2, while levels of the bax/caspase 3/caspase 9 proteins were decreased, which is consistent with inhibition of apoptosis.
Heart failure after myocardial infarction is a clinically common phenomenon. There is high morbidity and mortality following myocardial infarction, because the remaining heart tissue has to maintain adequate cardiac output, similar to the current management of congestive Fig. 4 Effects of miR-17-5p inhibition on the ERK pathway in injured cardiac tissue. After 7 or 28 days AMI, a mRNA expression of miR-17 was analyzed by real-time RT-PCR (n = 3, each group contains 5 hearts). b mRNA expression of ERK was analyzed by real-time RT-PCR (n = 3, each group contains 5 hearts). c Protein expression of ERK, P-ERK were examined by western blot. d ERK protein activity was evaluated by P-ERK/ERK (n = 3, each group contains 5 hearts) (*p < 0.05 vs. Ad-NC-Antagomir-17, # p < 0.05 vs. Ad-NC-Antagomir-17, n = 3) 1 3 heart failure (Kanashiro-Takeuchi et al. 2015). Our data showed that have shown that heart failure after myocardial infarction is closely related to the microcirculation damage of the heart (Teunissen et al. 2015). Heart function can be protected if microcirculation is improved after myocardial infarction, which would prevent symptoms of heart failure. Research has verified that inhibiting the expression of miR-17-5p can prevent endothelial cells from undergoing apoptosis and can also promote tube formation by vessel endothelial cells. Therefore, Fig. 5 Effects of miR-17-5p inhibition on apoptosis-related proteins in injured cardiac tissue. After 7 or 28 days AMI, a Western blot was used to detected apoptosis-related proteins. b Protein expression of anti-apoptosis protein bcl-2 was examined by western blot. c Levels of apoptosis protein Bax was measured by western blot. d Protein expression of apoptosis protein caspas3 was examined by western blot. e Relative protein levels of apoptosis protein caspas9 (n = 3, each group contains 5 hearts) (*p < 0.05 vs. Ad-NC-Antagomir-17, # p < 0.05 vs. Ad-NC-Antagomir-17, n = 3) MiR-17-5p silencing protects heart function after AMI by improving the microcirculation of the heart tissue, which is through decreasing the rate of apoptosis and repairing vascular injury. The effect of miR-17 on the heart is still not entirely clear, and many more potential mechanisms underlying this effect must be investigated.

Conclusion
Collectively, this study provided novel insights into the mechanism of miR-17-5p regulating endothelium apoptosis. Actually, apoptosis was inhibited by the Fig. 6 Effect of miR-17-5p inhibition on the histopathology in heart tissue after myocardial infarction at 7 or 28 days after the induction of AMI. a, b Representative hematoxylin and eosin-stained cardiac tissues from each experimental group (n = 5, light microscope, ×400). C, d Representative Masson trichrome-stained heart tissues. Collagen is stained blue (n = 5, light microscope, ×200) ( # p < 0.05 vs. *Ad-NC-Antagomir-17) 1 3 downregulation of miR-17-5p through activation of the ERK pathway. MiR-17-5p silencing protects heart function after AMI by improving the microcirculation of the heart tissue by decreasing the rate of apoptosis and repairing vascular injury. Fig. 7 Effect of miR-17-5p inhibition on the histopathology in heart tissue after myocardial infarction at 7 or 28 days after the induction of AMI. a, b Representative cardiac sections with immunohistochemical staining for CD31. Black arrows indicate areas of endothelial recovery, which are stained dark brown (n = 5, light microscope, ×400). c, d Representative TUNELstained cardiac sections. Black arrows indicate positive cells, which are stained dark brown (n = 5, light microscope, ×400) ( # p < 0.05 vs. *Ad-NC-Antagomir-17) Table 1 Effect of miR-17-5p inhibition on heart function after myocardial infarction *P < 0.05 which represent that the indicators (EF, FS, EDV, ESV, LVIDD and LVIDS) had significant differences between downregulation group (Ad-Aatagomir-17) and control group (Ad-NC-Aatagomir-17)