*4.3. Limitations of the Study*

There are several limitations to this study. An important limitation is the lack of functional measurements and investigation of vascular changes. The animal studies were designed to analyze cardiac but not vascular sequelae of hypertension. Unfortunately, only systolic BP values were measured. We are aware of the fact that diastolic BP would provide relevant data characterizing the vascular treatment effects. This limitation is because of the method of non-invasive BP measurement with a tail cuff device that did not allow measurement of diastolic BP. Data on TPR, which may reflect vascular changes, were only obtained from the ySHRs. In addition, we have compared changes in cardiac tissue with echocardiographic data from oSHRs reported previously [48].

Another limitation is in the analysis of collagen mRNA expression to characterize the development of fibrosis. Previous studies on rats stimulated with norepinephrine showed a good correlation between LV collagen mRNA expression and collagen amount in the histological specimen [50,51]. Fibrotic processes include enhanced collagen turnover and an increased collagen accumulation. We assume that mRNA expression does not adequately reflect the accumulation of collagen. Moreover, collagen mRNA and protein content develop with aging in a nonsynchronous way [46]. Collagen protein concentration or hydroxyproline content would more adequately reflect the intensity of fibrotic processes. We consider the histological degree of fibrosis to be a reliable and meaningful marker of fibrosis.

#### **5. Conclusions**

As elevated BP is the leading cause of premature death and is the major risk factor for a variety of cardiovascular disease events [3], early and effective antihypertensive therapy is of paramount importance for the patients. The present data of an animal study show that even a late-onset treatment of hypertension has both antihypertensive and cardioprotective effects. However, in those animals, SBP remained at significantly higher levels than in ySHRs, which received the same medication administered over a much shorter period of time at an early stage of life. Consequently, cardiac remodeling and transition into fibrosis were delayed but not prevented in the old animals. In contrast, fibrosis was significantly attenuated in ySHRs after only three weeks of therapy. This result gives strong reason to believe that a life-long antihypertensive therapy started at an early stage in life may prevent severe cardiac remodeling and transition into cardiac failure at advanced age.

The results emphasize that antihypertensive therapy should start as early as possible and be maintained as long as BP is elevated, usually life-long. Continuous BP control and strict adherence to treatment are inevitable. Early reduction in SBP, preferably to normotensive or near-normotensive values, contributes to prevent cardiac complications of hypertension.

**Author Contributions:** B.R., H.-G.Z., C.H. and J.B. contributed to the conception and design of the study. C.H., J.B. and B.R. performed the animal experiments including blood pressure measurements and the analyses on heart tissue. B.R. performed the statistics and created figures and tables. H.-G.Z. and B.R. drafted the original manuscript. All authors contributed to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This manuscript does not contain clinical studies or patient data. The study was conducted according to the guidelines of the Declaration of Helsinki. All animal protocols were approved by the state agency (Landesdirektion Sachsen, number and date of approval: TVV 36/08; 13 May 2009) in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and with the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes".

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors acknowledge support from the German Research Foundation (DFG) and Universität Leipzig within the program of Open Access Publishing.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**


#### **References**

