Peer review of: Targeting QKI-7 in vivo restores endothelial cell function in diabetes (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In general terms the paper by Yang et al proposes the finding which is novel that the RNA binding 
protein Quaking 7 is up-regulated within the diabetic conditions and when up-regulated by a likely 
splicing factor impairs endothelial cell function in a manner that is consistent with the mechanism 
of diabetes induced vascular injury. I suggest the authors consider the following 
suggesting/questions/areas for clarification. 
 
1) Is the increase in Quaking 7 expression that is seen following the iPSC differentiation and the 
resulting effects from high glucose “specific” for endothelia cells? Were parallel studies performed 
in other cell types derived from iPSC to determine the specificity of the observed findings. 
 
2) Studies done with varying concentrations should include an isoosmolar control with L-glucose 
being perhaps the best option though mannitol can also be used. If I missed this control, my 
apologies. 
 
3) I like the approach taken by the authors in regards to the identification of the splicing factor. 
While this approach could be viewed as too targeted, I think it is reasonable. What I would like to 
better understand is the magnitude of the genes that are differentially spliced by this factor? 
 
4) I like the studies on the cell-cell interaction protein expression being linked to function though I 
am less familiar with the system used vs. more conventional studies such as dextran permeability 
and electrical resistance, A bit more reference situation may be adequate here to limit this 
concern, 
 
5) While I appreciate the complexity of creating human derived iPSC the authors appear to be 
using an n=1 for the comparison of the human correlation of diabetes vs. no diabetes. 
 
6) The hind-limb ischemia data while consistent with the hypothesis also may need a bit more 
attention. More data on the specificity of the lentivirus for hitting endothelial cells vs. other cell 
types is needed. The general statement is that intramuscular delivery/injection of almost anything 
hits endothelial cells at a low frequency. This does tie back to one of the prior comments about 
specificity of the quaking 7 dysregulation in other cell types and the possibiliuty that some of the 
effects are from cells other than EC. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This interesting study investigates the role of QKI7 in diabetic vascular disease. In the first part, 
the authors used iPS-EC (exposed to high glucose) from mice or diabetic patients to evaluate the 
related signalling and function. They then assessed the potential targets and interactors and finally 
used in vivo models of ischemia where iPS-ECs expressing the protein were injected or the protein 
was silencing. 
The study provides novel evidence that QKI7 modulates vascular processes. The authors have 
used novel technologies in their study and the experimental work seems to be well performed and 
illustrated with details enough for reproduction. 
 
Major comments 
Although the cellular model is a sophisticated and certainly useful for screening, it would be 
certainly important to validate the upregulation of the RBP in endothelial cells and VSMCs from 
diabetic mice. Even more convincing would be to evaluate the RBP in leftover material from 
amputated limb of diabetic patients. Contact of potential collaborators providing such a material 

can be provided to the authors if they wish to test this in human samples. 
 
The source of human iPS should be clarified as they generically state are from type 1 and 2 
diabetic patients without specifying the line where the main results are shown. How do they 
explain the persistence of the RBP upregulation after iPS generation and endothelial cell 
differentiation? Did the high glucose increase the expression of the RBP in the human cells? 
Similarly, do commercial human endothelial cells exposed to high glucose increase the RBP 
expression? 
 
The in vivo model data are limited to the assessment of blood flow but there is apparently and 
surprisingly no data about vascular histology, including capillary density, arteriole density, state of 
myocytes etc so it is not possible to determine what is the structural basis of the perfusion effects 
and the cellular target of the injected factors. 
 
The conclusion of this being a therapeutic avenue is premature particularly in the light of the lack 
of experimental evidence in the in vivo studies showing only perfusion. 
 
It is also noted that authors used cells from diabetic donors type 1 and 2 or murine cells exposed 
to high glucose and then in vivo studies in type 1. High glucose is not the only factor responsible 
for diabetic complication, further osmotic controls are necessary. Diabetes type 1 and 2 are 
different pathologies, the authors should focus on one of the pathology and be consistent through 
the study in the use of a corresponding model. 
 
In conclusion, this is an interesting study but my enthusiasm is reduced by some flaws in the logic 
connection of the work, the overestimation of glucose induced effect in relation to a diabetic 
condition, the rationale to use derived cells instead of freshly isolated material and limitations of 
the in vivo study. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
The topic of the manuscript is clinically and scientifically significant. Microvascular dysfunction is a 
key process underlying the development of the cardiovascular complications of diabetes. In this 
manuscript the authors present a detailed study on the role of an isoform of the RNA binding 
protein Quaking in endothelial function and dysfunction in conditions of hyperglycemia. 
The authors report that when using induced pluripotent stem cell derived endothelial cells from 
mice are exposed to hyperglycemia the QKI-7 isoform is markedly upregulated and that the same 
holds true for untreated (hyperglycemia) human diabetes patient derived iPS-EC. 
Subsequent studies suggest that a shift in the balance of two potential RNA binding slicing factors 
drives the increase in QKI-7 and that the mechanism involves three genes involved in endothelial 
integrity, permeability and monocyte adhesion. When QKI-7 is suppressed in iPS-EC and these are 
infused in a murine ischemic model (and matrigel plugs) the cells promote “angiogenesis” 
The final conclusion is that QKI-7 plays a role in diabetic endothelial dysfunction and that the 
molecule could be a therapeutic tragic to counteract cardiovascular complications of this disease 
In most cells QKI-5 goes up first and than, at higher concentrations impacting on the splicing of its 
own transcript yielding more QKI-6 and 7. The impact of hyperglycemia of the relative regulation 
of the isoforms is an intriguing observation. 
 
 
 
Major comments 
 
While the conclusions drawn seem supported by the directions in the experiments in general the 
effects are moderate and it is often unclear how many (what is the n of the data?) and whether 

there are technical or biological duplicates. Also in several experiments it is unclear in which cells 
the experiments were performed. E.g. figure 5, 7D how many? Representative pictures? 
 
For instance, the conclusion can be drawn that human iPS-EC from diabetic patients express quite 
increased levels of QKI-7. Yet, this was shown for two iPS-EC lines from diabetes patients and two 
control human subjects. A true conclusion on the general applicability of this conclusion might 
need analysis of multiple cell lines. 
 
Also the source of the diabetes derived iPS-EC is not described other than “patients with type 1 or 
2 DM that has the disease over 15 years”. However, these diabetes types are quite different and 
can have an impact on the genetic background of the cells. Could this be more specified. 
 
The impact of hyperglycemia on the mouse cultures iPS-EC expression of QKI-7 is quite strong. It 
is somewhat unclear how hyperglycemia impacts on the RNA binding proteins CUG-BP and 
hnRNPM. What is the evidence that these RNA binding proteins physically bind to the QKI 
precursor RNA. It can not be excluded that overexpressing CUG-BP or hnRNPM affects genes 
upstream of QKI expression. Mutations for these RBPs in the QKI precursor could substantiate 
these conclusions. 
Ischemic hindlimb models usually mostly assess arteriogenesis much more than angiogenesis. 
These are different processes and should be taken into account 
 
 
Minor 
 
QKI-7 is known as a stabilizing factor on genes by binding to the 3’ UTR and poly A binding 
protein. Here QKI-7 is destabilizing the expression of three genes. This could be discussed. 
 
It is surprising that in figure 6C and D overexpression of a single gene that was down regulated by 
overexpression fully compensates for the loss of function. It can be assumed that multiple factor 
would be involved. Can this be explained. 
 
Would be informative to note in the introduction that QKI-7 is predominantly cytoplasmic..not 
directly involved in splicing. 
 
Figure 7A. How many plugs were analysed? Throughout the manuscript the n is problematic..and it 
is not defined what the n is biological vs technical controls