## 4 Discussion
### Cardiac LyV Density Is Related to Obesity  
The decrease in the LyV density in the Ang II-treated db/db mice compared to the controls but similar to the density in the db/db mice observed in our study suggests the significance of the environment associated with obesity and metabolic anomalies for vessel regression and/or impaired lymphangiogenesis. LyV regression may occur in this environment, as indicated by signs of injury observed in the LEC of the Ang II-treated db/db mice (Supplementary Figure S3A). In the literature, LyVs rarefaction in MetS has been shown in skin tissue in patients [84] and in obese mice, both with genetic and diet-induced obesity [85], suggesting the significance of obesity for LyV rarefaction. However, the dose and duration of Ang II administration, and other associated factors such as elevated plasma VEGF levels, are crucial for lymphangiogenesis. It has also been reported that Ang II infusion in mice leads not only to increased lymphangiogenesis and increased LyV permeability but also to myocardial edema, and these effects are dose- and time-dependent [16]. Similarly, [PERSON] et al. [86] observed that Ang II treatment for 6 weeks causes increased lymphangiogenesis, high VEGF expression and impaired lymph transport within lymphatics. Moreover, [PERSON] et al. have reported that Ang II, acting through the AT1 receptor, promotes the expression of lymphatic genes such as Lyve-1, Prox1, VEGF-C, and VEGFR and stimulates the proliferation and migration of LECs in vitro [15]. This type of LyV remodeling may also involve an altered balance between proliferation and death of LECsunder the influence of Ang II particularly, because obesity and other MetS comorbidities can accelerate apoptosis, including apoptosis of endothelial cells [87]. The relationship between the amount and remodeling of fibrotic tissue into more stiff collagen fibers and LyV rarefaction should be also taken into account, since the ECM provides a physical and biological environment for the formation and stabilization of the LyV network. It is known that the ECM mediates external mechanical signals into intracellular pathways via the cytoskeleton of the lymphatic endothelial cells, regulating LEC proliferation, vessel wall barrier tightness and other functions [39; 88]. Although we did not examine the ECM composition in detail, the TEM imaging allowed us to confirm the presence of numerous thick collagen I fibers, in particular in the Ang II-treated db/db mice, whose presence is associated with an increased myocardial stiffness [89]. In addition, the increased tension of the ECM and cardiac wall may contribute to damage or apoptosis of the LECs [90]. Moreover, the BM, a component of the ECM, becomes significantly thickened in cardiac LyVs of db/db and Ang II-treated db/db mice compared to controls (Figure 10C). Since the major molecules of BM are collagen IV (Coll IV) and laminin [91; 92], we presume that the BM thickness also relates to the increase in the structural stiffness of LyVs. This is especially true in relation of reports on Coll IV being considered an \"inelastic\" molecule [92; 93; 94]. Our results concerning the thickened BM in the Ang II-treated db/db mice are in line with the study by [PERSON], who demonstrated a significantly increased level of mRNA for Coll IV in the myocardium of Ang II-treated db/db mice [52]. In addition, the BM determines the distribution of AnchFs, since AnchFs are visible only on the \"bare\" cell membrane and play a role in the recruitment of growth factors and interactions with cell receptors; the BM components also regulate cell proliferation [95]. The abnormal structure and disruption of the BM may be caused by pathological MAPK activation [96]. MAPK activation triggered by Ang II binding to the AT1 receptor stimulates multiple downstream pathways important for the progression of vascular remodeling, hypertension and other cardiovascular diseases (e.g., atherosclerosis) [97].