Blood pressure changes PVAT function and transcriptome: use of the mid-thoracic aorta coarcted rat

Abstract

Perivascular adipose tissue (PVAT) modifies the contractile function of the vessel it surrounds (outside-in signaling). Little work points to the vessel actively affecting its surrounding PVAT. We hypothesized that inside-out arterial signaling to PVAT would be evidenced by the response of PVAT to changes in tangential vascular wall stress. Rats coarcted in the mid-thoracic aorta created PVAT tissues that would exemplify pressure-dependent changes (above vs. below coarctation); a sham rat was used as a control. Radiotelemetry revealed a ∼20 mmHg systolic pressure gradient across the coarctation 4 wk after surgery. Four measures (histochemical, adipocyte progenitor proliferation and differentiation, isometric tone, and bulk mRNA sequencing) were used to compare PVAT above versus below the ligature in sham and coarcted rats. Neither aortic collagen deposition in PVAT nor arterial media/radius ratio above coarctation was increased versus below segments. However, differentiated adipocytes derived from PVAT above the coarctation accumulated substantially less triglycerides versus those below; their relative proliferation rate as adipogenic precursors was not different. Functionally, the ability of PVAT to assist stress relaxation of isolated aorta was reduced in rings above versus below the coarctation. Transcriptomic analyses revealed that the coarctation resulted in more differentially expressed genes (DEGs) between PVAT above versus below when compared with sham samples from the same locations. A majority of DEGs were in PVAT below the coarctation and were enriched in neuronal/synaptic terms. These findings provide initial evidence that signaling from the vascular wall, as stimulated by a pressure change, influences the function and transcriptional profile of its PVAT.NEW & NOTEWORTHY A mid-thoracic aorta coarcted rat was created to generate a stable pressure difference above versus below the coarctation ligature. This study determined that the PVAT around the thoracic aorta exposed to a higher pressure has a significantly reduced ability to assist stress relaxation versus that below the ligature and appears to retain the ability to be anticontractile. At the same time, the PVAT around the thoracic aorta exposed to higher pressure had a reduced adipogenic potential versus that below the ligature. Transcriptomics analyses indicated that PVAT below the coarctation showed the greatest number of DEGs with an increased profile of the synaptic neurotransmitter gene network.

Keywords: PVAT; adipocyte; mechanotransduction.

Fig. 1.

A: photograph and diagram of ligature placement on the descending thoracic aorta of the rat. B: radiotelemetric measures of mean (top) and systolic/diastolic (bottom) pressures in coarcted male Sprague–Dawley rats for 1 mo after implantation of a dual radiotelemeter (carotid for pressure above the ligature, femoral for pressure below). Points are means + SE for N number of individual rats. *Significant differences between bracketed groups as determined by mixed-effect analysis.

Fig. 2.

A, left: area of PVAT between sham vs. coarcted male Sprague–Dawley rats from above to below the ligature. Right: quantitation of medial thickness and arterial radius, reported as a ratio, for rings from above and below the ligature both in sham (left: two bars) and coarcted (right: two bars) rats. Bars are means + SE for the number of animals depicted by each of the symbols scattered around the means. Two sham and two coarcted rats had incomplete ring structures, which made the measures unobtainable. B, left: image of picrosirius red (PSR) staining in thoracic aortic sections immediately above and below the coarctation, taken from male rats a month after surgery. Representative of seven different animals. Right: quantitation of PSR staining in thoracic aortic PVAT above (open bar) and below (gray bar) the ligature; individual symbols are values for 5–6 male rats. C, left: perilipin1 (PLN1) staining of aorta from coarcted and sham rats from above or below the ligature. Red represents a signal greater than the baseline threshold determined by the corresponding no primary tissue section. Representative of five rats. Right: quantification of the PLN1 signal as a percent of PVAT area from sham vs. coarcted animals from above and below the ligature. D: distribution of lipid droplet areas in white (left) and brown (right) PVAT above (A) and below (B) the ligature in coarcted (CO, n = 6) and sham (SH, n = 5) male Sprague–Dawley rats. Bars with different letters or * differ significantly (ab, *P < 0.05). PVAT, perivascular adipose tissue.

Fig. 3.

A: proliferation of PVAT adipocyte progenitor cells (AP) above (open bar) and below (gray bar) the coarctation from sham (left, n = 4) and coarcted (right, n = 10) male Sprague–Dawley rats at 8, 48, and 96 h of culture. Points are values for individual rats around bars representing means + SE. B, top: representative images of AdipoRed staining in AP from above (left) and below (right) the coarctation in sham and coarcted male Sprague–Dawley rats after 14 days of culture in adipogenic media. Bottom: quantitation of TAG (triglyceride accumulation as adipored RFU) in sham (left, n = 3) or coarcted (right, n = 8) AP from aortic PVAT that was above (open bar) or below (gray bar) the ligature. Points are values for individual rats around bars representing means + SE. *P < 0.05, for one-way ANOVA followed by Tukey’s multiple comparisons. C: gene expression of adipogenic markers by RT qPCR in sham (left, n = 4) or coarcted (right, n = 4) AP from aortic PVAT that was above (open bar) or below (gray bar) the ligature after 14 days of culture in adipogenic media. Values are relative mRNA abundance after normalization with the reference gene Rps29. Significant differences are indicated by letters a, b, c, and d (P < 0.05). PVAT, perivascular adipose tissue; RFU, .

Fig. 4.

A: representative tracing of stress relaxation in the isolated rat thoracic aorta as assisted by PVAT. PVAT = tissues without PVAT; +PVAT = tissues with PVAT intact around aorta. B: cumulative tension achieved with each stretch step vs. no relaxation (gray starred line) comparing responses of tissues from above and below the ligature in sham (left) and coarcted (right) male Sprague–Dawley rats. Points represent means ± SE for the number of animals in parentheses. *Significant differences in maximums as determined by a Student’s t test (P < 0.05). C: the response of vessels stretched in B to a maximum concentration of PE (10 5 M) at each stretch step from sham (left) and coarcted (right) male rats. Points represent means ± SE for the number of animals in parentheses. PVAT, perivascular adipose tissue.

Fig. 5.

A: principal components analysis of the transcriptomics data. B: few DEGs (differentially expressed genes) are identified comparing coarcted and sham rats. Coarctation (D) induces more DEGs than in the sham rats (C). Red dots are statistically significant DEGs. E: comparison of the Log2fold change of the three DEG sets: sham unique, shared, and coarcted unique. The expressions (F) and coarctation effect index (shaded blue bars) of the three DEG sets in both sham and coarcted rats. CAB, coarcted above; CBE, coarcted below; SAB, sham above; SBE, sham below. The coarctation effect index defined as Log2FC (coarcted) − Log2FC (sham). The expression (numbers) is log transformed read counts. PVAT tissues are from coarcted (n = 6) and sham (n = 5) male Sprague–Dawley rats. PVAT, perivascular adipose tissue.

Fig. 6.

The three DEG sets enriched ten clusters of Gene Ontology Biological Process (GO) terms. A: enriched GO terms in the three DEG sets were color-coded by −log10 (P value) and ten coherent clusters (1–10) were identified. The number of enriched terms for each DEG set is labeled at the right side of the bar. The order of ten clusters are aligned with that in B. The order of terms in each cluster is ranked based on the Euclidian distance of the P value. B: clustered networks of enriched GO terms for each DEG set. Each node is a GO term and it is color-coded as the DEG sets enriched in this term. Blue, green, and light blue colors represent the sham unique, shared, and coarcted unique DEG sets, respectively. C: representative DEGs having the highest coarctation effect index value in each cluster. The numbers at upper left corners of each plot are aligned with those in B. CAB, coarcted above; CBE, coarcted below; SAB, sham above; SBE, sham below. Effect: coarctation effective index. PVAT tissues are from coarcted (n = 6) and sham (n = 5) male Sprague–Dawley rats. DEG, differentially expressed gene; PVAT, perivascular adipose tissue.

Fig. 7.

Rendering/synthesis of data presented within this manuscript. Grey shade,  perivascular adipose tissue total area; APC, adipocyte progenitor cell.